Tumor antigens BFA4 and BCY1 for prevention and / or treatment of cancer

ABSTRACT

The present invention relates to a nucleic acid encoding a polypeptide and the use of the nucleic acid or polypeptide in preventing and/or treating cancer. In particular, the invention relates to improved vectors for the insertion and expression of foreign genes encoding tumor antigens for use in immunotherapeutic treatment of cancer.

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Ser. Nos. 60/394,346filed Jul. 3, 2002; 60/394,503 filed Jul. 9, 2002; 60/411,833 filed Sep.18, 2002; and, 60/445,342 filed Feb. 6, 2003, all of which being herebyincorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to a nucleic acid encoding apolypeptide and the use of the nucleic acid or polypeptide in preventingand/or treating cancer. In particular, the invention relates to improvedvectors for the insertion and expression of foreign genes encoding tumorantigens for use in immunotherapeutic treatment of cancer.

BACKGROUND OF THE INVENTION

[0003] There has been tremendous increase in last few years in thedevelopment of cancer vaccines with Tumour-associated antigens (TAAs)due to the great advances in identification of molecules based on theexpression profiling on primary tumours and normal cells with the helpof several techniques such as high density microarray, SEREX,immunohistochemistry (IHC), RT-PCR, in-situ hybridization (ISH) andlaser capture microscopy (Rosenberg, Immunity, 1999; Sgroi et al, 1999,Schena et al, 1995, Offringa et al, 2000). The TAAs are antigensexpressed or over-expressed by tumour cells and could be specific to oneor several tumours for example CEA antigen is expressed in colorectal,breast and lung cancers. Sgroi et al (1999) identified several genesdifferentially expressed in invasive and metastatic carcinoma cells withcombined use of laser capture microdissection and cDNA microarrays.Several delivery systems like DNA or viruses could be used fortherapeutic vaccination against human cancers (Bonnet et al, 2000) andcan elicit immune responses and also break immune tolerance againstTAAs. Tumour cells can be rendered more immunogenic by insertingtransgenes encoding T cell co-stimulatory molecules such as B7.1 orcytokines such as IFN-γ, IL2, or GM-CSF, among others. Co-expression ofa TAA and a cytokine or a co-stimulatory molecule can develop effectivetherapeutic vaccine (Hodge et al, 95, Bronte et al, 1995, Chamberlain etal, 1996).

[0004] There is a need in the art for reagents and methodologies usefulin stimulating an immune response to prevent or treat cancers. Thepresent invention provides such reagents and methodologies whichovercome many of the difficulties encountered by others in attempting totreat cancer.

SUMMARY OF THE INVENTION

[0005] The present invention provides an immunogenic target foradministration to a patient to prevent and/or treat cancer. Inparticular, the immunogenic target is a tumor antigen (“TA”) and/or anangiogenesis-associated antigen (“AA”). In one embodiment, theimmunogenic target is encoded by SEQ ID NO.: 1 or 3 or has the aminoacid sequence of SEQ ID NO.: 2 or 4. In certain embodiments, the TAand/or AA are administered to a patient as a nucleic acid containedwithin a plasmid or other delivery vector, such as a recombinant virus.The TA and/or AA may also be administered in combination with an immunestimulator, such as a co-stimulatory molecule or adjuvant.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006]FIG. 1. BFA4 cDNA sequence.

[0007]FIG. 2. BFA4 amino acid sequence.

[0008]FIG. 3. BCY1 nucleotide (A) and amino acid (B) sequences.

DETAILED DESCRIPTION

[0009] The present invention provides reagents and methodologies usefulfor treating and/or preventing cancer. All references cited within thisapplication are incorporated by reference.

[0010] In one embodiment, the present invention relates to the inductionor enhancement of an immune response against one or more tumor antigens(“TA”) to prevent and/or treat cancer. In certain embodiments, one ormore TAs may be combined. In preferred embodiments, the immune responseresults from expression of a TA in a host cell following administrationof a nucleic acid vector encoding the tumor antigen or the tumor antigenitself in the form of a peptide or polypeptide, for example.

[0011] As used herein, an “antigen” is a molecule (such as apolypeptide) or a portion thereof that produces an immune response in ahost to whom the antigen has been administered. The immune response mayinclude the production of antibodies that bind to at least one epitopeof the antigen and/or the generation of a cellular immune responseagainst cells expressing an epitope of the antigen. The response may bean enhancement of a current immune response by, for example, causingincreased antibody production, production of antibodies with increasedaffinity for the antigen, or an increased cellular response (i.e.,increased T cells). An antigen that produces an immune response mayalternatively be referred to as being immunogenic or as an immunogen. Indescribing the present invention, a TA may be referred to as an“immunogenic target”.

[0012] TA includes both tumor-associated antigens (TAAs) andtumor-specific antigens (TSAs), where a cancerous cell is the source ofthe antigen. A TAA is an antigen that is expressed on the surface of atumor cell in higher amounts than is observed on normal cells or anantigen that is expressed on normal cells during fetal development. ATSA is an antigen that is unique to tumor cells and is not expressed onnormal cells. TA further includes TAAs or TSAs, antigenic fragmentsthereof, and modified versions that retain their antigenicity.

[0013] TAs are typically classified into five categories according totheir expression pattern, function, or genetic origin: cancer-testis(CT) antigens (i.e., MAGE, NY-ESO-1); melanocyte differentiationantigens (i.e., Melan A/MART-1, tyrosinase, gp100); mutational antigens(i.e., MUM-1, p53, CDK-4); overexpressed ‘self’ antigens (i.e.,HER-2/neu, p53); and, viral antigens (i.e., HPV, EBV). For the purposesof practicing the present invention, a suitable TA is any TA thatinduces or enhances an anti-tumor immune response in a host to whom theTA has been administered. Suitable TAs include, for example, gp100 (Coxet al., Science, 264:716-719 (1994)), MART-1/Melan A (Kawakami et al.,J. Exp. Med., 180:347-352 (1994)), gp75 (TRP-1) (Wang et al., J. Exp.Med., 186:1131-1140 (1996)), tyrosinase (Wolfel et al., Eur. J Immunol.,24:759-764 (1994); WO 200175117; WO 200175016; WO 200175007), NY-ESO-1(WO 98/14464; WO 99/18206), melanoma proteoglycan (Hellstrom et al., J.Immunol., 130:1467-1472 (1983)), MAGE family antigens (i.e., MAGE-1,2,3,4,6,12, 51; Van der Bruggen et al., Science, 254:1643-1647 (1991);U.S. Pat. No. 6,235,525; CN 1319611), BAGE family antigens (Boel et al.,Immunity, 2:167-175 (1995)), GAGE family antigens (i.e., GAGE-1,2; Vanden Eynde et al., J. Exp. Med., 182:689-698 (1995); U.S. Pat. No.6,013,765), RAGE family antigens (i.e., RAGE-1; Gaugler et at.,Immunogenetics, 44:323-330 (1996); U.S. Pat. No. 5,939,526),N-acetylglucosaminyltransferase-V (Guilloux et at., J. Exp. Med.,183:1173-1183 (1996)), p15 (Robbins et al., J. Immunol. 154:5944-5950(1995)), β-catenin (Robbins et al., J. Exp. Med., 183:1185-1192 (1996)),MUM-1 (Coulie et al., Proc. Natl. Acad. Sci. USA, 92:7976-7980 (1995)),cyclin dependent kinase-4 (CDK4) (Wolfel et al., Science, 269:1281-1284(1995)), p21-ras (Fossum et at., Int. J Cancer, 56:40-45 (1994)),BCR-abl (Bocchia et al., Blood, 85:2680-2684 (1995)), p53 (Theobald etal., Proc. Natl. Acad. Sci. USA, 92:11993-11997 (1995)), p185 HER2/neu(erb-B1; Fisk et al., J. Exp. Med., 181:2109-2117 (1995)), epidermalgrowth factor receptor (EGFR) (Harris et al., Breast Cancer Res. Treat,29:1-2 (1994)), carcinoembryonic antigens (CEA) (Kwong et al., J. Natl.Cancer Inst., 85:982-990 (1995) U.S. Pat. Nos. 5,756,103; 5,274,087;5,571,710; 6,071,716; 5,698,530; 6,045,802; EP 263933; EP 346710; and,EP 784483); carcinoma-associated mutated mucins (i.e., MUC-1 geneproducts; Jerome et al., J. Immunol., 151:1654-1662 (1993)); EBNA geneproducts of EBV (i.e., EBNA-1; Rickinson et al., Cancer Surveys,13:53-80 (1992)); E7, E6 proteins of human papillomavirus (Ressing etal., J. Immunol, 154:5934-5943 (1995)); prostate specific antigen (PSA;Xue et al., The Prostate, 30:73-78 (1997)); prostate specific membraneantigen (PSMA; Israeli, et al., Cancer Res., 54:1807-1811 (1994));idiotypic epitopes or antigens, for example, immunoglobulin idiotypes orT cell receptor idiotypes (Chen et al., J. Immunol., 153:4775-4787(1994)); KSA (U.S. Pat. No. 5,348,887), kinesin 2 (Dietz, et al. BiochemBiophys Res Commun 2000 Sep. 7;275(3):731-8), HIP-55, TGFβ-1anti-apoptotic factor (Toomey, et al. Br J Biomed Sci2001;58(3):177-83), tumor protein D52 (Bryne J. A., et al., Genomics,35:523-532 (1996)), H1FT, NY-BR-1 (WO 01/47959), NY-BR-62, NY-BR-75,NY-BR-85, NY-BR-87, NY-BR-96 (Scanlan, M. Serologic and BioinformaticApproaches to the Identification of Human Tumor Antigens, in CancerVaccines 2000, Cancer Research Institute, New York, N.Y.), BFA4 (SEQ IDNOS.: 26 and 27), or BCY1 (SEQ ID NOS.: 28 and 29), including“wild-type” (i.e., normally encoded by the genome, naturally-occurring),modified, and mutated versions as well as other fragments andderivatives thereof. Any of these TAs may be utilized alone or incombination with one another in a co-immunization protocol.

[0014] In certain cases, it may be beneficial to co-immunize patientswith both TA and other antigens, such as angiogenesis-associatedantigens (“AA”). An AA is an immunogenic molecule (i.e., peptide,polypeptide) associated with cells involved in the induction and/orcontinued development of blood vessels. For example, an AA may beexpressed on an endothelial cell (“EC”), which is a primary structuralcomponent of blood vessels. Where the cancer is cancer, it is preferredthat that the AA be found within or near blood vessels that supply atumor. Immunization of a patient against an AA preferably results in ananti-AA immune response whereby angiogenic processes that occur near orwithin tumors are prevented and/or inhibited.

[0015] Exemplary AAs include, for example, vascular endothelial growthfactor (i.e., VEGF; Bemardini, et al. J. Urol., 2001, 166(4): 1275-9;Stames, et al. J. Thorac. Cardiovasc. Surg., 2001, 122(3): 518-23; Dias,et al. Blood, 2002, 99: 2179-2184), the VEGF receptor (i.e., VEGF-R,flk-1/KDR; Starnes, et al. J. Thorac. Cardiovasc. Surg., 2001, 122(3):518-23), EPH receptors (i.e., EPHA2; Gerety, et al. 1999, Cell, 4:403-414), epidermal growth factor receptor (i.e., EGFR; Ciardeillo, etal. Clin. Cancer Res., 2001, 7(10): 2958-70), basic fibroblast growthfactor (i.e., bFGF; Davidson, et al. Clin. Exp. Metastasis 2000,18(6):501-7; Poon, et al. Am J. Surg., 2001, 182(3):298-304), platelet-derivedcell growth factor (i.e., PDGF-B), platelet-derived endothelial cellgrowth factor (PD-ECGF; Hong, et al. J. Mol. Med., 2001, 8(2):141-8),transforming growth factors (i.e., TGF-α; Hong, et al. J. Mol. Med.,2001, 8(2):141-8), endoglin, (Balza, et al. Int. J. Cancer, 2001, 94:579-585), Id proteins (Benezra, R. Trends Cardiovasc. Med., 2001,11(6):237-41), proteases such as uPA, uPAR, and matrixmetalloproteinases (MMP-2, MMP-9; Djonov, et al. J. Pathol., 2001,195(2):147-55), nitric oxide synthase (Am. J. Ophthalmol., 2001,132(4):551-6), aminopeptidase (Rouslhati, E. Nature Cancer, 2: 84-90,2002), thrombospondins (i.e., TSP-1, TSP-2; Alvarez, et al. Gynecol.Oncol., 2001, 82(2):273-8; Seki, et al. Int. J. Oncol., 2001,19(2):305-10), k-ras (Zhang, et al. Cancer Res., 2001, 61(16):6050-4),Wnt (Zhang, et al. Cancer Res., 2001, 61(16):6050-4), cyclin-dependentkinases (CDKs; Drug Resist. Updat. 2000, 3(2):83-88), microtubules(Timar, et al. 2001. Path. Oncol. Res., 7(2): 85-94), heat shockproteins (i.e., HSP90 (Timar, supra)), heparin-binding factors (i.e.,heparinase; Gohji, et al. Int. J. Cancer, 2001, 95(5):295-301),synthases (i.e., ATP synthase, thymidilate synthase), collagenreceptors, integrins (i.e., ανβ3, ανβ5, α1β1, α2β1, α5β1), or surfaceproteolglycan NG2, among others, including “wild-type” (i.e., normallyencoded by the genome, naturally-occurring), modified, mutated versionsas well as other fragments and derivatives thereof. Any of these targetsmay be suitable in practicing the present invention, either alone or incombination with one another or with other agents.

[0016] In certain embodiments, a nucleic acid molecule encoding animmunogenic target is utilized. The nucleic acid molecule may compriseor consist of a nucleotide sequence encoding one or more immunogenictargets, or fragments or derivatives thereof, such as that contained ina DNA insert in an ATCC Deposit. The term “nucleic acid sequence” or“nucleic acid molecule” refers to a DNA or RNA sequence. The termencompasses molecules formed from any of the known base analogs of DNAand RNA such as, but not limited to 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinyl-cytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxy-methylaminomethyluracil, dihydrouracil, inosine,N6-iso-pentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonyl-methyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine, among others.

[0017] An isolated nucleic acid molecule is one that: (1) is separatedfrom at least about 50 percent of proteins, lipids, carbohydrates, orother materials with which it is naturally found when total nucleic acidis isolated from the source cells; (2) is not be linked to all or aportion of a polynucleotide to which the nucleic acid molecule is linkedin nature; (3) is operably linked to a polynucleotide which it is notlinked to in nature; and/or, (4) does not occur in nature as part of alarger polynucleotide sequence. Preferably, the isolated nucleic acidmolecule of the present invention is substantially free from any othercontaminating nucleic acid molecule(s) or other contaminants that arefound in its natural environment that would interfere with its use inpolypeptide production or its therapeutic, diagnostic, prophylactic orresearch use. As used herein, the term “naturally occurring” or “native”or “naturally found” when used in connection with biological materialssuch as nucleic acid molecules, polypeptides, host cells, and the like,refers to materials which are found in nature and are not manipulated byman. Similarly, “non-naturally occurring” or “non-native” as used hereinrefers to a material that is not found in nature or that has beenstructurally modified or synthesized by man.

[0018] The identity of two or more nucleic acid or polypeptide moleculesis determined by comparing the sequences. As known in the art,“identity” means the degree of sequence relatedness between nucleic acidmolecules or polypeptides as determined by the match between the unitsmaking up the molecules (i.e., nucleotides or amino acid residues).Identity measures the percent of identical matches between the smallerof two or more sequences with gap alignments (if any) addressed by aparticular mathematical model or computer program (i.e., an algorithm).Identity between nucleic acid sequences may also be determined by theability of the related sequence to hybridize to the nucleic acidsequence or isolated nucleic acid molecule. In defining such sequences,the term “highly stringent conditions” and “moderately stringentconditions” refer to procedures that permit hybridization of nucleicacid strands whose sequences are complementary, and to excludehybridization of significantly mismatched nucleic acids. Examples of“highly stringent conditions” for hybridization and washing are 0.015 Msodium chloride, 0.0015 M sodium citrate at 65-68° C. or 0.015 M sodiumchloride, 0.0015 M sodium citrate, and 50% formamide at 42° C. (see, forexample, Sambrook, Fritsch & Maniatis, Molecular Cloning: A LaboratoryManual (2nd ed., Cold Spring Harbor Laboratory, 1989); Anderson et al.,Nucleic Acid Hybridisation: A Practical Approach Ch. 4 (IRL PressLimited)). The term “moderately stringent conditions” refers toconditions under which a DNA duplex with a greater degree of base pairmismatching than could occur under “highly stringent conditions” is ableto form. Exemplary moderately stringent conditions are 0.015 M sodiumchloride, 0.0015 M sodium citrate at 50-65° C. or 0.015 M sodiumchloride, 0.0015 M sodium citrate, and 20% formamide at 37-50° C. By wayof example, moderately stringent conditions of 50° C. in 0.015 M sodiumion will allow about a 21% mismatch. During hybridization, other agentsmay be included in the hybridization and washing buffers for the purposeof reducing non-specific and/or background hybridization. Examples are0.1% bovine serum albumin, 0.1% polyvinyl-pyrrolidone, 0.1% sodiumpyrophosphate, 0.1% sodium dodecylsulfate, NaDodSO₄, (SDS), ficoll,Denhardt's solution, sonicated salmon sperm DNA (or anothernon-complementary DNA), and dextran sulfate, although other suitableagents can also be used. The concentration and types of these additivescan be changed without substantially affecting the stringency of thehybridization conditions. Hybridization experiments are usually carriedout at pH 6.8-7.4; however, at typical ionic strength conditions, therate of hybridization is nearly independent of pH.

[0019] In preferred embodiments of the present invention, vectors areused to transfer a nucleic acid sequence encoding a polypeptide to acell. A vector is any molecule used to transfer a nucleic acid sequenceto a host cell. In certain cases, an expression vector is utilized. Anexpression vector is a nucleic acid molecule that is suitable fortransformation of a host cell and contains nucleic acid sequences thatdirect and/or control the expression of the transferred nucleic acidsequences. Expression includes, but is not limited to, processes such astranscription, translation, and splicing, if introns are present.Expression vectors typically comprise one or more flanking sequencesoperably linked to a heterologous nucleic acid sequence encoding apolypeptide. Flanking sequences may be homologous (i.e., from the samespecies and/or strain as the host cell), heterologous (i.e., from aspecies other than the host cell species or strain), hybrid (i.e., acombination of flanking sequences from more than one source), orsynthetic, for example.

[0020] A flanking sequence is preferably capable of effecting thereplication, transcription and/or translation of the coding sequence andis operably linked to a coding sequence. As used herein, the termoperably linked refers to a linkage of polynucleotide elements in afunctional relationship. For instance, a promoter or enhancer isoperably linked to a coding sequence if it affects the transcription ofthe coding sequence. However, a flanking sequence need not necessarilybe contiguous with the coding sequence, so long as it functionscorrectly. Thus, for example, intervening untranslated yet transcribedsequences can be present between a promoter sequence and the codingsequence and the promoter sequence may still be considered operablylinked to the coding sequence. Similarly, an enhancer sequence may belocated upstream or downstream from the coding sequence and affecttranscription of the sequence.

[0021] In certain embodiments, it is preferred that the flankingsequence is a trascriptional regulatory region that drives high-levelgene expression in the target cell. The transcriptional regulatoryregion may comprise, for example, a promoter, enhancer, silencer,repressor element, or combinations thereof. The transcriptionalregulatory region may be either constitutive, tissue-specific, cell-typespecific (i.e., the region is drives higher levels of transcription in aone type of tissue or cell as compared to another), or regulatable(i.e., responsive to interaction with a compound such as tetracycline).The source of a transcriptional regulatory region may be any prokaryoticor eukaryotic organism, any vertebrate or invertebrate organism, or anyplant, provided that the flanking sequence functions in a cell bycausing transcription of a nucleic acid within that cell. A wide varietyof transcriptional regulatory regions may be utilized in practicing thepresent invention.

[0022] Suitable transcriptional regulatory regions include the CMVpromoter (i.e., the CMV-immediate early promoter); promoters fromeukaryotic genes (i.e., the estrogen-inducible chicken ovalbumin gene,the interferon genes, the gluco-corticoid-inducible tyrosineaminotransferase gene, and the thymidine kinase gene); and the majorearly and late adenovirus gene promoters; the SV40 early promoter region(Bemoist and Chambon, 1981, Nature 290:304-10); the promoter containedin the 3′ long terminal repeat (LTR) of Rous sarcoma virus (RSV)(Yamamoto, et al., 1980, Cell 22:787-97); the herpes simplex virusthymidine kinase (HSV-TK) promoter (Wagner et al., 1981, Proc. Natl.Acad. Sci. U.S.A. 78:1444-45); the regulatory sequences of themetallothionine gene (Brinster et al., 1982, Nature 296:39-42);prokaryotic expression vectors such as the beta-lactamase promoter(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A.,75:3727-31); or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad.Sci. US.A., 80:21-25). Tissue- and/or cell-type specific transcriptionalcontrol regions include, for example, the elastase I gene control regionwhich is active in pancreatic acinar cells (Swift et al., 1984, Cell38:639-46; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol.50:399-409 (1986); MacDonald, 1987, Hepatology 7:425-515); the insulingene control region which is active in pancreatic beta cells (Hanahan,1985, Nature 315:115-22); the immunoglobulin gene control region whichis active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-58;Adames et al., 1985, Nature 318:533-38; Alexander et al., 1987, Mol.Cell. Biol., 7:1436-44); the mouse mammary tumor virus control region intesticular, breast, lymphoid and mast cells (Leder et al., 1986, Cell45:485-95); the albumin gene control region in liver (Pinkert et al.,1987, Genes and Devel. 1:268-76); the alpha-feto-protein gene controlregion in liver (Krumlauf et al., 1985, Mol. Cell. Biol., 5:1639-48;Hammer et al., 1987, Science 235:53-58); the alpha 1-antitrypsin genecontrol region in liver (Kelsey et al., 1987, Genes and Devel.1:161-71); the beta-globin gene control region in myeloid cells (Mogramet al., 1985, Nature 315:338-40; Kollias et al., 1986, Cell 46:89-94);the myelin basic protein gene control region in oligodendrocyte cells inthe brain (Readhead et al., 1987, Cell 48:703-12); the myosin lightchain-2 gene control region in skeletal muscle (Sani, 1985, Nature314:283-86); the gonadotropic releasing hormone gene control region inthe hypothalamus (Mason et al., 1986, Science 234:1372-78), and thetyrosinase promoter in melanoma cells (Hart, I. Semin Oncol 1996February;23(1):154-8; Siders, et al. Cancer Gene Ther 1998September-October;5(5):281-91), among others. Inducible promoters thatare activated in the presence of a certain compound or condition such aslight, heat, radiation, tetracycline, or heat shock proteins, forexample, may also be utilized (see, for example, WO 00/10612). Othersuitable promoters are known in the art.

[0023] As described above, enhancers may also be suitable flankingsequences. Enhancers are cis-acting elements of DNA, usually about10-300 bp in length, that act on the promoter to increase transcription.Enhancers are typically orientation- and position-independent, havingbeen identified both 5′ and 3′ to controlled coding sequences. Severalenhancer sequences available from mammalian genes are known (i.e.,globin, elastase, albumin, alpha-feto-protein and insulin). Similarly,the SV40 enhancer, the cytomegalovirus early promoter enhancer, thepolyoma enhancer, and adenovirus enhancers are useful with eukaryoticpromoter sequences. While an enhancer may be spliced into the vector ata position 5′ or 3′ to nucleic acid coding sequence, it is typicallylocated at a site 5′ from the promoter. Other suitable enhancers areknown in the art, and would be applicable to the present invention.

[0024] While preparing reagents of the present invention, cells may needto be transfected or transformed. Transfection refers to the uptake offoreign or exogenous DNA by a cell, and a cell has been transfected whenthe exogenous DNA has been introduced inside the cell membrane. A numberof transfection techniques are well known in the art (i.e., Graham etal., 1973, Virology 52:456; Sambrook et al., Molecular Cloning, ALaboratory Manual (Cold Spring Harbor Laboratories, 1989); Davis et al.,Basic Methods in Molecular Biology (Elsevier, 1986); and Chu et al.,1981, Gene 13:197). Such techniques can be used to introduce one or moreexogenous DNA moieties into suitable host cells.

[0025] In certain embodiments, it is preferred that transfection of acell results in transformation of that cell. A cell is transformed whenthere is a change in a characteristic of the cell, being transformedwhen it has been modified to contain a new nucleic acid. Followingtransfection, the transfected nucleic acid may recombine with that ofthe cell by physically integrating into a chromosome of the cell, may bemaintained transiently as an episomal element without being replicated,or may replicate independently as a plasmid. A cell is stablytransformed when the nucleic acid is replicated with the division of thecell.

[0026] The present invention further provides isolated immunogenictargets in polypeptide form. A polypeptide is considered isolated whereit: (1) has been separated from at least about 50 percent ofpolynucleotides, lipids, carbohydrates, or other materials with which itis naturally found when isolated from the source cell; (2) is not linked(by covalent or noncovalent interaction) to all or a portion of apolypeptide to which the “isolated polypeptide” is linked in nature; (3)is operably linked (by covalent or noncovalent interaction) to apolypeptide with which it is not linked in nature; or, (4) does notoccur in nature. Preferably, the isolated polypeptide is substantiallyfree from any other contaminating polypeptides or other contaminantsthat are found in its natural environment that would interfere with itstherapeutic, diagnostic, prophylactic or research use.

[0027] Immunogenic target polypeptides may be mature polypeptides, asdefined herein, and may or may not have an amino terminal methionineresidue, depending on the method by which they are prepared. Furthercontemplated are related polypeptides such as, for example, fragments,variants (i.e., allelic, splice), orthologs, homologues, andderivatives, for example, that possess at least one characteristic oractivity (i.e., activity, antigenicity) of the immunogenic target. Alsorelated are peptides, which refers to a series of contiguous amino acidresidues having a sequence corresponding to at least a portion of thepolypeptide from which its sequence is derived. In preferredembodiments, the peptide comprises about 5-10 amino acids, 10-15 aminoacids, 15-20 amino acids, 20-30 amino acids, or 30-50 amino acids. In amore preferred embodiment, a peptide comprises 9-12 amino acids,suitable for presentation upon Class I MHC molecules, for example.

[0028] A fragment of a nucleic acid or polypeptide comprises atruncation of the sequence (i.e., nucleic acid or polypeptide) at theamino terminus (with or without a leader sequence) and/or the carboxyterminus. Fragments may also include variants (i.e., allelic, splice),orthologs, homologues, and other variants having one or more amino acidadditions or substitutions or internal deletions as compared to theparental sequence. In preferred embodiments, truncations and/ordeletions comprise about 10 amino acids, 20 amino acids, 30 amino acids,40 amino acids, 50 amino acids, or more. The polypeptide fragments soproduced will comprise about 10 amino acids, 25 amino acids, 30 aminoacids, 40 amino acids, 50 amino acids, 60 amino acids, 70 amino acids,or more. Such polypeptide fragments may optionally comprise an aminoterminal methionine residue. It will be appreciated that such fragmentscan be used, for example, to generate antibodies or cellular immuneresponses to immunogenic target polypeptides.

[0029] A variant is a sequence having one or more sequencesubstitutions, deletions, and/or additions as compared to the subjectsequence. Variants may be naturally occurring or artificiallyconstructed. Such variants may be prepared from the correspondingnucleic acid molecules. In preferred embodiments, the variants have from1 to 3, or from 1 to 5, or from 1 to 10, or from 1 to 15, or from 1 to20, or from 1 to 25, or from 1 to 30, or from 1 to 40, or from 1 to 50,or more than 50 amino acid substitutions, insertions, additions and/ordeletions.

[0030] An allelic variant is one of several possible naturally-occurringalternate forms of a gene occupying a given locus on a chromosome of anorganism or a population of organisms. A splice variant is a polypeptidegenerated from one of several RNA transcript resulting from splicing ofa primary transcript. An ortholog is a similar nucleic acid orpolypeptide sequence from another species. For example, the mouse andhuman versions of an immunogenic target polypeptide may be consideredorthologs of each other. A derivative of a sequence is one that isderived from a parental sequence those sequences having substitutions,additions, deletions, or chemically modified variants. Variants may alsoinclude fusion proteins, which refers to the fusion of one or more firstsequences (such as a peptide) at the amino or carboxy terminus of atleast one other sequence (such as a heterologous peptide).

[0031] “Similarity” is a concept related to identity, except thatsimilarity refers to a measure of relatedness which includes bothidentical matches and conservative substitution matches. If twopolypeptide sequences have, for example, 10/20 identical amino acids,and the remainder are all non-conservative substitutions, then thepercent identity and similarity would both be 50%. If in the sameexample, there are five more positions where there are conservativesubstitutions, then the percent identity remains 50%, but the percentsimilarity would be 75% (15/20). Therefore, in cases where there areconservative substitutions, the percent similarity between twopolypeptides will be higher than the percent identity between those twopolypeptides.

[0032] Substitutions may be conservative, or non-conservative, or anycombination thereof. Conservative amino acid modifications to thesequence of a polypeptide (and the corresponding modifications to theencoding nucleotides) may produce polypeptides having functional andchemical characteristics similar to those of a parental polypeptide. Forexample, a “conservative amino acid substitution” may involve asubstitution of a native amino acid residue with a non-native residuesuch that there is little or no effect on the size, polarity, charge,hydrophobicity, or hydrophilicity of the amino acid residue at thatposition and, in particlar, does not result in decreased immunogenicity.Suitable conservative amino acid substitutions are shown in Table I.TABLE I Original Preferred Residues Exemplary SubstitutionsSubstitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln GlnAsp Glu Glu Cys Ser, Ala Ser Gln Asn Asn Glu Asp Asp Gly Pro, Ala AlaHis Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe, Norleucine LeuLeu Norleucine, Ile, Val, Met, Ala, Phe Ile Lys Arg, 1,4 Diamino-butyricAcid, Gln, Asn Arg Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Tyr LeuPro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp,Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Ala, Norleucine Leu

[0033] A skilled artisan will be able to determine suitable variants ofpolypeptide using well-known techniques. For identifying suitable areasof the molecule that may be changed without destroying biologicalactivity (i.e., MHC binding, immunogenicity), one skilled in the art maytarget areas not believed to be important for that activity. Forexample, when similar polypeptides with similar activities from the samespecies or from other species are known, one skilled in the art maycompare the amino acid sequence of a polypeptide to such similarpolypeptides. By performing such analyses, one can identify residues andportions of the molecules that are conserved among similar polypeptides.It will be appreciated that changes in areas of the molecule that arenot conserved relative to such similar polypeptides would be less likelyto adversely affect the biological activity and/or structure of apolypeptide. Similarly, the residues required for binding to MHC areknown, and may be modified to improve binding. However, modificationsresulting in decreased binding to MHC will not be appropriate in mostsituations. One skilled in the art would also know that, even inrelatively conserved regions, one may substitute chemically similaramino acids for the naturally occurring residues while retainingactivity. Therefore, even areas that may be important for biologicalactivity or for structure may be subject to conservative amino acidsubstitutions without destroying the biological activity or withoutadversely affecting the polypeptide structure.

[0034] Other preferred polypeptide variants include glycosylationvariants wherein the number and/or type of glycosylation sites have beenaltered compared to the subject amino acid sequence. In one embodiment,polypeptide variants comprise a greater or a lesser number of N-linkedglycosylation sites than the subject amino acid sequence. An N-linkedglycosylation site is characterized by the sequence Asn-X-Ser orAsn-X-Thr, wherein the amino acid residue designated as X may be anyamino acid residue except proline. The substitution of amino acidresidues to create this sequence provides a potential new site for theaddition of an N-linked carbohydrate chain. Alternatively, substitutionsthat eliminate this sequence will remove an existing N-linkedcarbohydrate chain. Also provided is a rearrangement of N-linkedcarbohydrate chains wherein one or more N-linked glycosylation sites(typically those that are naturally occurring) are eliminated and one ormore new N-linked sites are created. To affect O-linked glycosylation ofa polypeptide, one would modify serine and/or threonine residues.

[0035] Additional preferred variants include cysteine variants, whereinone or more cysteine residues are deleted or substituted with anotheramino acid (e.g., serine) as compared to the subject amino acid sequenceset. Cysteine variants are useful when polypeptides must be refoldedinto a biologically active conformation such as after the isolation ofinsoluble inclusion bodies. Cysteine variants generally have fewercysteine residues than the native protein, and typically have an evennumber to minimize interactions resulting from unpaired cysteines.

[0036] In other embodiments, the isolated polypeptides of the currentinvention include fusion polypeptide segments that assist inpurification of the polypeptides. Fusions can be made either at theamino terminus or at the carboxy terminus of the subject polypeptidevariant thereof. Fusions may be direct with no linker or adaptermolecule or may be through a linker or adapter molecule. A linker oradapter molecule may be one or more amino acid residues, typically fromabout 20 to about 50 amino acid residues. A linker or adapter moleculemay also be designed with a cleavage site for a DNA restrictionendonuclease or for a protease to allow for the separation of the fusedmoieties. It will be appreciated that once constructed, the fusionpolypeptides can be derivatized according to the methods describedherein. Suitable fusion segments include, among others, metal bindingdomains (e.g., a poly-histidine segment), immunoglobulin binding domains(i.e., Protein A, Protein G, T cell, B cell, Fc receptor, or complementprotein antibody-binding domains), sugar binding domains (e.g., amaltose binding domain), and/or a “tag” domain (i.e., at least a portionof α-galactosidase, a strep tag peptide, a T7 tag peptide, a FLAGpeptide, or other domains that can be purified using compounds that bindto the domain, such as monoclonal antibodies). This tag is typicallyfused to the polypeptide upon expression of the polypeptide, and canserve as a means for affinity purification of the sequence of interestpolypeptide from the host cell. Affinity purification can beaccomplished, for example, by column chromatography using antibodiesagainst the tag as an affinity matrix. Optionally, the tag cansubsequently be removed from the purified sequence of interestpolypeptide by various means such as using certain peptidases forcleavage. As described below, fusions may also be made between a TA anda co-stimulatory components such as the chemokines CXC10 (IP-10), CCL7(MCP-3), or CCL5 (RANTES), for example.

[0037] A fusion motif may enhance transport of an immunogenic target toan MHC processing compartment, such as the endoplasmic reticulum. Thesesequences, referred to as tranduction or transcytosis sequences, includesequences derived from HIV tat (see Kim et al. 1997 J. Immunol.159:1666), Drosophila antennapedia (see Schutze-Redelmeier et al. 1996J. Immunol. 157:650), or human period-1 protein (hPER1; in particular,SRRHHCRSKAKRSRHH).

[0038] In addition, the polypeptide or variant thereof may be fused to ahomologous polypeptide to form a homodimer or to a heterologouspolypeptide to form a heterodimer. Heterologous peptides andpolypeptides include, but are not limited to: an epitope to allow forthe detection and/or isolation of a fusion polypeptide; a transmembranereceptor protein or a portion thereof, such as an extracellular domainor a transmembrane and intracellular domain; a ligand or a portionthereof which binds to a transmembrane receptor protein; an enzyme orportion thereof which is catalytically active; a polypeptide or peptidewhich promotes oligomerization, such as a leucine zipper domain; apolypeptide or peptide which increases stability, such as animmunoglobulin constant region; and a polypeptide which has atherapeutic activity different from the polypeptide or variant thereof.

[0039] In certain embodiments, it may be advantageous to combine anucleic acid sequence encoding an immunogenic target, polypeptide, orderivative thereof with one or more co-stimulatory component(s) such ascell surface proteins, cytokines or chemokines in a composition of thepresent invention. The co-stimulatory component may be included in thecomposition as a polypeptide or as a nucleic acid encoding thepolypeptide, for example. Suitable co-stimulatory molecules include, forinstance, polypeptides that bind members of the CD28 family (i.e., CD28,ICOS; Hutloff, et al. Nature 1999, 397: 263-265; Peach, et al. J Exp Med1994, 180: 2049-2058) such as the CD28 binding polypeptides B7.1 (CD80;Schwartz, 1992; Chen et al, 1992; Ellis, et al. J. Immunol., 156(8):2700-9) and B7.2 (CD86; Ellis, et al. J. Immunol., 156(8): 2700-9);polypeptides which bind members of the integrin family (i.e., LFA-1(CD11a/CD18); Sedwick, et al. J Immunol 1999, 162: 1367-1375; Wülfing,et al. Science 1998, 282: 2266-2269; Lub, et al. Immunol Today 1995, 16:479-483) including members of the ICAM family (i.e., ICAM-1, -2 or -3);polypeptides which bind CD2 family members (i.e., CD2, signallinglymphocyte activation molecule (CDw150 or “SLAM”; Aversa, et al. JImmunol 1997, 158: 4036-4044)) such as CD58 (LFA-3; CD2 ligand; Davis,et al. Immunol Today 1996, 17: 177-187) or SLAM ligands (Sayos, et al.Nature 1998, 395: 462469); polypeptides which bind heat stable antigen(HSA or CD24; Zhou, et al. Eur J Immunol 1997, 27: 2524-2528);polypeptides which bind to members of the TNF receptor (TNFR) family(i.e., 4-1BB (CD137; Vinay, et al. Semin Immunol 1998, 10: 481-489),OX40 (CD134; Weinberg, et al. Semin Immunol 1998, 10: 471-480; Higgins,et al. J Immunol 1999, 162: 486-493), and CD27 (Lens, et al. SeminImmunol 1998, 10: 491-499)) such as 4-1BBL (4-1BB ligand; Vinay, et al.Semin Immunol 1998, 10: 481-48; DeBenedette, et al. J Immunol 1997, 158:551-559), TNFR associated factor-1 (TRAF-1; 4-1BB ligand; Saoulli, etal. J Exp Med 1998, 187: 1849-1862, Arch, et al. Mol Cell Biol 1998, 18:558-565), TRAF-2 (4-1BB and OX40 ligand; Saoulli, et al. J Exp Med 1998,187: 1849-1862; Oshima, et al. Int Immunol 1998, 10: 517-526, Kawamata,et al. J Biol Chem 1998, 273: 5808-5814), TRAF-3 (4-1BB and OX40 ligand;Arch, et al. Mol Cell Biol 1998, 18: 558-565; Jang, et al. BiochemBiophys Res Commun 1998, 242: 613-620; Kawamata S, et al. J Biol Chem1998, 273: 5808-5814), OX40L (OX40 ligand; Gramaglia, et al. J Immunol1998, 161: 6510-6517), TRAF-5 (OX40 ligand; Arch, et al. Mol Cell Biol1998, 18: 558-565; Kawamata, et al. J Biol Chem 1998, 273: 5808-5814),and CD70 (CD27 ligand; Couderc, et al. Cancer Gene Ther., 5(3): 163-75).CD154 (CD40 ligand or “CD40L”; Gurunathan, et al. J. Immunol., 1998,161: 4563-4571; Sine, et al. Hum. Gene Ther., 2001, 12: 1091-1102) mayalso be suitable.

[0040] One or more cytokines may also be suitable co-stimulatorycomponents or “adjuvants”, either as polypeptides or being encoded bynucleic acids contained within the compositions of the present invention(Parmiani, et al. Immunol Lett 2000 Sep. 15; 74(1): 41-4; Berzofsky, etal.

[0041] Nature Immunol. 1: 209-219). Suitable cytokines include, forexample, interleukin-2 (IL-2) (Rosenberg, et al. Nature Med. 4: 321-327(1998)), IL-4, IL-7, IL-12 (reviewed by Pardoll, 1992; Harries, et al.J. Gene Med. 2000 July-August;2(4):243-9; Rao, et al. J. Immunol. 156:3357-3365 (1996)), IL-15 (Xin, et al. Vaccine, 17:858-866, 1999), IL-16(Cruikshank, et al. J. Leuk Biol. 67(6): 757-66, 2000), IL-18 (J. CancerRes. Clin. Oncol. 2001. 127(12): 718-726), GM-CSF (CSF (Disis, et al.Blood, 88: 202-210 (1996)), tumor necrosis factor-alpha (TNF-α), orinterferons such as IFN-α or INF-γ. Other cytokines may also be suitablefor practicing the present invention, as is known in the art.

[0042] Chemokines may also be utilized. For example, fusion proteinscomprising CXCL10 (IP-10) and CCL7 (MCP-3) fused to a tumor self-antigenhave been shown to induce anti-tumor, immunity (Biragyn, et al. NatureBiotech. 1999, 17: 253-258). The chemokines CCL3 (MIP-1α) and CCL5(RANTES) (Boyer, et al. Vaccine, 1999, 17 (Supp. 2): S53-S64) may alsobe of use in practicing the present invention. Other suitable chemokinesare known in the art.

[0043] It is also known in the art that suppressive or negativeregulatory immune mechanisms may be blocked, resulting in enhancedimmune responses. For instance, treatment with anti-CTLA-4 (Shrikant, etal. Immunity, 1996, 14: 145-155; Sutmuller, et al. J. Exp. Med., 2001,20 194: 823-832), anti-CD25 (Sutmuller, supra), anti-CD4 (Matsui, et al.J. Immunol., 1999, 163:

[0044]184-193), the fusion protein IL13Ra2-Fc (Terabe, et al. NatureImmunol., 2000, 1: 515-520), and combinations thereof (i.e., anti-CTLA-4and anti-CD25, Sutmuller, supra) have been shown to upregulateanti-tumor immune responses and would be suitable in practicing thepresent invention.

[0045] Any of these components may be used alone or in combination withother agents. For instance, it has been shown that a combination ofCD80, ICAM-1 and LFA-3 (“TRICOM”) may potentiate anti-cancer immuneresponses (Hodge, et al. Cancer Res. 59: 5800-5807 (1999). Othereffective combinations include, for example, IL-12+GM-CSF (Ahlers, etal. J. Immunol., 158: 3947-3958 (1997); Iwasaki, et al. J. Immunol. 158:4591-4601 (1997)), IL-12+GM-CSF+TNF-α (Ahlers, et al. Int Immunol. 13:897-908 (2001)), CD80+IL-12 (Fruend, et al. Int. J Cancer, 85: 508-517(2000); Rao, et al. supra), and CD86+GM-CSF+IL-12 (Iwasaki, supra). Oneof skill in the art would be aware of additional combinations useful incarrying out the present invention.In addition, the skilled artisanwould be aware of additional reagents or methods that may be used tomodulate such mechanisms. These reagents and methods, as well as othersknown by those of skill in the art, may be utilized in practicing thepresent invention.

[0046] Additional strategies for improving the efficiency of nucleicacid-based immunization may also be used including, for example, the useof self-replicating viral replicons (Caley, et al. 1999. Vaccine, 17:3124-2135; Dubensky, et al. 2000. Mol. Med. 6: 723-732; Leitner, et al.2000. Cancer Res. 60: 51-55), codon optimization (Liu, et al. 2000. Mol.Ther., 1: 497-500; Dubensky, supra; Huang, et al. 2001. J Virol. 75:4947-4951), in vivo electroporation (Widera, et al. 2000. J. Immunol.164: 4635-3640), incorporation of CpG stimulatory motifs (Gurunathan, etal. Ann. Rev. Immunol., 2000, 18: 927-974; Leitner, supra; Cho, et al.J. Immunol. 168(10):4907-13), sequences for targeting of the endocyticor ubiquitin-processing pathways (Thomson, et al. 1998. J. Virol. 72:2246-2252; Velders, et al. 2001. J. Immunol. 166: 5366-5373), Marek'sdisease virus type 1 VP22 sequences (J. Virol. 76(6):2676-82, 2002),prime-boost regimens (Gurunathan, supra; Sullivan, et al. 2000. Nature,408: 605-609; Hanke, et al. 1998. Vaccine, 16: 439-445; Amara, et al.2001. Science, 292: 69-74), and the use of mucosal delivery vectors suchas Salmonella (Darji, et al. 1997. Cell, 91: 765-775; Woo, et al. 2001.Vaccine, 19: 2945-2954). Other methods are known in the art, some ofwhich are described below.

[0047] Chemotherapeutic agents, radiation, anti-angiogenic compounds, orother agents may also be utilized in treating and/or preventing cancerusing immunogenic targets (Sebti, et al. Oncogene 2000 Dec.27;19(56):6566-73). For example, in treating metastatic breast cancer,useful chemotherapeutic agents include cyclophosphamide, doxorubicin,paclitaxel, docetaxel, navelbine, capecitabine, and mitomycin C, amongothers. Combination chemotherapeutic regimens have also proven effectiveincluding cyclophosphamide+methotrexate+5-fluorouracil;cyclophosphamide+doxorubicin+5-fluorouracil; or,cyclophosphamide+doxorubicin, for example. Other compounds such asprednisone, a taxane, navelbine, mitomycin C, or vinblastine have beenutlized for various reasons. A majority of breast cancer patients haveestrogen-receptor positive (ER+) tumors and in these patients, endocrinetherapy (i.e., tamoxifen) is preferred over chemotherapy. For suchpatients, tamoxifen or, as a second line therapy, progestins(medroxyprogesterone acetate or megestrol acetate) are preferred.Aromatase inhibitors (i.e., aminoglutethimide and analogs thereof suchas letrozole) decrease the availability of estrogen needed to maintaintumor growth and may be used as second or third line endocrine therapyin certain patients.

[0048] Other cancers may require different chemotherapeutic regimens.For example, metastatic colorectal cancer is typically treated withCamptosar (irinotecan or CPT-11), 5-fluorouracil or leucovorin, alone orin combination with one another. Proteinase and integrin inhibitors suchas as the MMP inhibitors marimastate (British Biotech), COL-3(Collagenex), Neovastat (Aeterna), AG3340 (Agouron), BMS-275291 (BristolMyers Squibb), CGS 27023A (Novartis) or the integrin inhibitors Vitaxin(Medimmune), or MED1522 (Merck KgaA) may also be suitable for use. Assuch, immunological targeting of immunogenic targets associated withcolorectal cancer could be performed in combination with a treatmentusing those chemotherapeutic agents. Similarly, chemotherapeutic agentsused to treat other types of cancers are well-known in the art and maybe combined with the immunogenic targets described herein.

[0049] Many anti-angiogenic agents are known in the art and would besuitable for co-administration with the immunogenic target vaccines(see, for example, Timar, et al. 2001. Pathology Oncol. Res., 7(2):85-94). Such agents include, for example, physiological agents such asgrowth factors (i.e., ANG-2, NK1,2,4 (HGF), transforming growth factorbeta (TGF-β)), cytokines (i.e., interferons such as IFN-α, -β, -γ,platelet factor 4 (PF-4), PR-39), proteases (i.e., cleaved AT-III,collagen XVIII fragment (Endostatin)), HmwKallikrein-d5 plasmin fragment(Angiostatin), prothrombin-F1-2, TSP-1), protease inhibitors (i.e.,tissue inhibitor of metalloproteases such as TIMP-1, -2, or -3; maspin;plasminogen activator-inhibitors such as PAI-1; pigment epitheliumderived factor (PEDF)), Tumstatin (available through ILEX, Inc.),antibody products (i.e., the collagen-binding antibodies HUIV26, HU177,XL313; anti-VEGF; anti-integrin (i.e., Vitaxin, (Lxsys))), andglycosidases (i.e., heparinase-I, -III). “Chemical” or modifiedphysiological agents known or believed to have anti-angiogenic potentialinclude, for example, vinblastine, taxol, ketoconazole, thalidomide,dolestatin, combrestatin A, rapamycin (Guba, et al. 2002, Nature Med.,8: 128-135), CEP-7055 (available from Cephalon, Inc.), flavone aceticacid, Bay 12-9566 (Bayer Corp.), AG3340 (Agouron, Inc.), CGS 27023A(Novartis), tetracylcine derivatives (i.e., COL-3 (Collagenix, Inc.)),Neovastat (Aeterna), BMS-275291 (Bristol-Myers Squibb), low dose 5-FU,low dose methotrexate (MTX), irsofladine, radicicol, cyclosporine,captopril, celecoxib, D45152-sulphated polysaccharide, cationic protein(Protamine), cationic peptide-VEGF, Suramin (polysulphonated napthylurea), compounds that interfere with the function or production of VEGF(i.e., SU5416 or SU6668 (Sugen), PTK787/ZK22584 (Novartis)), DistamycinA, Angiozyme (ribozyme), isoflavinoids, staurosporine derivatives,genistein, EMD121974 (Merck KcgaA), tyrphostins, isoquinolones, retinoicacid, carboxyamidotriazole, TNP-470, octreotide, 2-methoxyestradiol,aminosterols (i.e., squalamine), glutathione analogues (i.e.,N-acteyl-L-cysteine), combretastatin A-4 (Oxigene), Eph receptorblocking agents (Nature, 414:933-938, 2001), Rh-Angiostatin,Rh-Endostatin (WO 01/93897), cyclic-RGD peptide, accutin-disintegrin,benzodiazepenes, humanized anti-avb3 Ab, Rh-PAI-2, amiloride,p-amidobenzamidine, anti-uPA ab, anti-uPAR Ab,L-phanylalanin-N-methylamides (i.e., Batimistat, Marimastat), AG3340,and minocycline. Many other suitable agents are known in the art andwould suffice in practicing the present invention.

[0050] The present invention may also be utilized in combination with“non-traditional” methods of treating cancer. For example, it hasrecently been demonstrated that administration of certain anaerobicbacteria may assist in slowing tumor growth. In one study, Clostridiumnovyi was modified to eliminate a toxin gene carried on a phage episomeand administered to mice with colorectal tumors (Dang, et al. P.N.A.S.USA, 98(26): 15155-15160, 2001). In combination with chemotherapy, thetreatment was shown to cause tumor necrosis in the animals. The reagentsand methodologies described in this application may be combined withsuch treatment methodologies.

[0051] Nucleic acids encoding immunogenic targets may be administered topatients by any of several available techniques. Various viral vectorsthat have been successfully utilized for introducing a nucleic acid to ahost include retrovirus, adenovirus, adeno-associated virus (AAV),herpes virus, and poxvirus, among others. It is understood in the artthat many such viral vectors are available in the art. The vectors ofthe present invention may be constructed using standard recombinanttechniques widely available to one skilled in the art. Such techniquesmay be found in common molecular biology references such as MolecularCloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring HarborLaboratory Press), Gene Expression Technology (Methods in Enzymology,Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego,Calif.), and PCR Protocols: A Guide to Methods and Applications (Innis,et al. 1990. Academic Press, San Diego, Calif.).

[0052] Preferred retroviral vectors are derivatives of lentivirus aswell as derivatives of murine or avian retroviruses. Examples ofsuitable retroviral vectors include, for example, Moloney murineleukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murinemammary tumor virus (MuMTV), SIV, BIV, HIV and Rous Sarcoma Virus (RSV).A number of retroviral vectors can incorporate multiple exogenousnucleic acid sequences. As recombinant retroviruses are defective, theyrequire assistance in order to produce infectious vector particles. Thisassistance can be provided by, for example, helper cell lines encodingretrovirus structural genes. Suitable helper cell lines include T2,PA317 and PA12, among others. The vector virions produced using suchcell lines may then be used to infect a tissue cell line, such as NIH3T3 cells, to produce large quantities of chimeric retroviral virions.Retroviral vectors may be administered by traditional methods (i.e.,injection) or by implantation of a “producer cell line” in proximity tothe target cell population (Culver, K., et al., 1994, Hum. Gene Ther., 5(3): 343-79; Culver, K., et al., Cold Spring Harb. Symp. Quant. Biol.,59: 685-90); Oldfield, E., 1993, Hum. Gene Ther., 4 (1): 39-69). Theproducer cell line is engineered to produce a viral vector and releasesviral particles in the vicinity of the target cell. A portion of thereleased viral particles contact the target cells and infect thosecells, thus delivering a nucleic acid of the present invention to thetarget cell. Following infection of the target cell, expression of thenucleic acid of the vector occurs.

[0053] Adenoviral vectors have proven especially useful for genetransfer into eukaryotic cells (Rosenfeld, M., et al., 1991, Science,252 (5004): 431-4; Crystal, R., et al., 1994, Nat. Genet., 8 (1):42-51), the study eukaryotic gene expression (Levrero, M., et al., 1991,Gene, 101 (2): 195-202), vaccine development (Graham, F. and Prevec, L.,1992, Biotechnology, 20: 363-90), and in animal models(Stratford-Perricaudet, L., et al., 1992, Bone Marrow Transplant., 9(Suppl. 1): 151-2 ; Rich, D., et al., 1993, Hum. Gene Ther., 4 (4):461-76). Experimental routes for administrating recombinant Ad todifferent tissues in vivo have included intratracheal instillation(Rosenfeld, M., et al., 1992, Cell, 68 (1): 143-55) injection intomuscle (Quantin, B., et al., 1992, Proc. Natl. Acad. Sci. U.S.A., 89(7): 2581-4), peripheral intravenous injection (Herz, J., and Gerard,R., 1993, Proc. Natl. Acad. Sci. U.S.A., 90 (7): 2812-6) andstereotactic inoculation to brain (Le Gal La Salle, G., et al., 1993,Science, 259 (5097): 988-90), among others.

[0054] Adeno-associated virus (AAV) demonstrates high-level infectivity,broad host range and specificity in integrating into the host cellgenome (Hermonat, P., et al., 1984, Proc. Natl. Acad. Sci. U.S.A., 81(20): 6466-70). And Herpes Simplex Virus type-1 (HSV-1) is yet anotherattractive vector system, especially for use in the nervous systembecause of its neurotropic property (Geller, A., et al., 1991, TrendsNeurosci., 14 (10): 428-32; Glorioso, et al., 1995, Mol. Biotechnol., 4(1): 87-99; Glorioso, et al., 1995, Annu. Rev. Microbiol., 49: 675-710).

[0055] Poxvirus is another useful expression vector (Smith, et al. 1983,Gene, 25 (1): 21-8; Moss, et al, 1992, Biotechnology, 20: 345-62; Moss,et al, 1992, Curr. Top. Microbiol. Immunol., 158: 25-38; Moss, et al.1991. Science, 252: 1662-1667). Poxviruses shown to be useful includevaccinia, NYVAC, avipox, fowlpox, canarypox, ALVAC, and ALVAC(2), amongothers.

[0056] NYVAC (vP866) was derived from the Copenhagen vaccine strain ofvaccinia virus by deleting six nonessential regions of the genomeencoding known or potential virulence factors (see, for example, U.S.Pat. Nos. 5,364,773 and 5,494,807). The deletion loci were alsoengineered as recipient loci for the insertion of foreign genes. Thedeleted regions are: thymidine kinase gene (TK; J2R); hemorrhagic region(u; B13R+B14R); A type inclusion body region (ATI; A26L); hemagglutiningene (HA; A56R); host range gene region (C7L-K1L); and, large subunit,ribonucleotide reductase (14L). NYVAC is a genetically engineeredvaccinia virus strain that was generated by the specific deletion ofeighteen open reading frames encoding gene products associated withvirulence and host range. NYVAC has been show to be useful forexpressing TAs (see, for example, U.S. Pat. No. 6,265,189). NYVAC(vP866), vP994, vCP205, vCP1433, placZH6H4Lreverse, pMPC6H6K3E3 andpC3H6FHVB were also deposited with the ATCC under the terms of theBudapest Treaty, accession numbers VR-2559, VR-2558, VR-2557, VR-2556,ATCC-97913, ATCC-97912, and ATCC-97914, respectively.

[0057] ALVAC-based recombinant viruses (i.e., ALVAC-1 and ALVAC-2) arealso suitable for use in practicing the present invention (see, forexample, U.S. Pat. No. 5,756,103). ALVAC(2) is identical to ALVAC(1)except that ALVAC(2) genome comprises the vaccinia E3L and K3L genesunder the control of vaccinia promoters (U.S. Pat. No. 6,130,066;Beattie et al., 1995a, 1995b, 1991; Chang et al., 1992; Davies et al.,1993). Both ALVAC(1) and ALVAC(2) have been demonstrated to be useful inexpressing foreign DNA sequences, such as TAs (Tartaglia et al., 1993a,b; U.S. Pat. No. 5,833,975). ALVAC was deposited under the terms ofthe Budapest Treaty with the American Type Culture Collection (ATCC),10801 University Boulevard, Manassas, Va. 20110-2209, USA, ATCCaccession number VR-2547.

[0058] Another useful poxvirus vector is TROVAC. TROVAC refers to anattenuated fowlpox that was a plaque-cloned isolate derived from theFP-1 vaccine strain of fowlpoxvirus which is licensed for vaccination of1 day old chicks. TROVAC was likewise deposited under the terms of theBudapest Treaty with the ATCC, accession number 2553.

[0059] “Non-viral” plasmid vectors may also be suitable in practicingthe present invention. Preferred plasmid vectors are compatible withbacterial, insect, and/or mammalian host cells. Such vectors include,for example, PCR-II, pCR3, and pcDNA3.1 (Invitrogen, San Diego, Calif.),pBSII (Stratagene, La Jolla, Calif.), pET15 (Novagen, Madison, Wis.),pGEX (Pharmacia Biotech, Piscataway, N.J.), pEGFP-N2 (Clontech, PaloAlto, Calif.), pETL (BlueBachl, Invitrogen), pDSR-alpha (PCT pub. No. WO90/14363) and pFastBacDual (Gibco-BRL, Grand Island, N.Y.) as well asBluescript® plasmid derivatives (a high copy number COLE1-basedphagemid, Stratagene Cloning Systems, La Jolla, Calif.), PCR cloningplasmids designed for cloning Taq-amplified PCR products (e.g., TOPO™ TAcloning® kit, PCR2.1® plasmid derivatives, Invitrogen, Carlsbad,Calif.). Bacterial vectors may also be used with the current invention.These vectors include, for example, Shigella, Salmonella, Vibriocholerae, Lactobacillus, Bacille calmette guérin (BCG), andStreptococcus (see for example, WO 88/6626; WO 90/0594; WO 91/13157; WO92/1796; and WO 92/21376). Many other non-viral plasmid expressionvectors and systems are known in the art and could be used with thecurrent invention.

[0060] Suitable nucleic acid delivery techniques include DNA-ligandcomplexes, adenovirus-ligand-DNA complexes, direct injection of DNA,CaPO₄ precipitation, gene gun techniques, electroporation, and colloidaldispersion systems, among others. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome, which are artificial membrane vesicles usefulas delivery vehicles in vitro and in vivo. RNA, DNA and intact virionscan be encapsulated within the aqueous interior and be delivered tocells in a biologically active form (Fraley, R., et al., 1981, TrendsBiochem. Sci., 6: 77). The composition of the liposome is usually acombination of phospholipids, particularlyhigh-phase-transition-temperature phospholipids, usually in combinationwith steroids, especially cholesterol. Other phospholipids or otherlipids may also be used. The physical characteristics of liposomesdepend on pH, ionic strength, and the presence of divalent cations.Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

[0061] An immunogenic target may also be administered in combinationwith one or more adjuvants to boost the immune response. Exemplaryadjuvants are shown in Table II below: TABLE II Types of ImmunologicAdjuvants Type of Adjuvant General Examples Specific Examples/ReferencesGel-type Aluminum hydroxide/phosphate (Aggerbeck and Heron, 1995) (“alumadjuvants”) Calcium phosphate (Relyveld, 1986) Microbial Muramyldipeptide (MDP) (Chedid et al., 1986) Bacterial exotoxins Cholera toxin(CT), E. coli labile toxin (LT)(Freytag and Clements, 1999)Endotoxin-based adjuvants Monophosphoryl lipid A (MPL) (Ulrich andMyers, 1995) Other bacterial CpG oligonucleotides (Corral and Petray,2000), BCG sequences (Krieg, et al. Nature, 374: 576), tetanus toxoid(Rice, et al. J. Immunol., 2001, 167: 1558-1565) ParticulateBiodegradable (Gupta et al., 1998) Polymer microspheresImmunostimulatory complexes (Morein and Bengtsson, 1999) (ISCOMs)Liposomes (Wassef et al., 1994) Oil-emulsion Freund's incompleteadjuvant (Jensen et al., 1998) and Microfluidized emulsions MF59 (Ott etal., 1995) surfactant- SAF (Allison and Byars, 1992) based (Allison,1999) adjuvants Saponins QS-21 (Kensil, 1996) Synthetic Muramyl peptidederivatives Murabutide (Lederer, 1986) Threony-MDP (Allison, 1997)Nonionic block copolymers L121 (Allison, 1999) Polyphosphazene (PCPP)(Payne et al., 1995) Synthetic polynucleotides Poly A:U, Poly I:C(Johnson, 1994) Thalidomide derivatives CC-4047/ACTIMID (J. Immunol.,168(10): 4914-9)

[0062] The immunogenic targets of the present invention may also be usedto generate antibodies for use in screening assays or for immunotherapy.Other uses would be apparent to one of skill in the art. The term“antibody” includes antibody fragments, as are known in the art,including Fab, Fab₂, single chain antibodies (Fv for example), humanizedantibodies, chimeric antibodies, human antibodies, produced by severalmethods as are known in the art. Methods of preparing and utilizingvarious types of antibodies are well-known to those of skill in the artand would be suitable in practicing the present invention (see, forexample, Harlow, et al. Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, 1988; Harlow, et al. Using Antibodies: A LaboratoryManual, Portable Protocol No. 1, 1998; Kohler and Milstein, Nature,256:495 (1975)); Jones et al. Nature, 321:522-525 (1986); Riechmann etal. Nature, 332:323-329 (1988); Presta (Curr. Op. Struct. Biol.,2:593-596 (1992); Verhoeyen et al. (Science, 239:1534-1536 (1988);Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol.Biol., 222:581 (1991); Cole et al., Monoclonal Antibodies and CancerTherapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol.,147(1):86-95 (1991); Marks et al., Bio/Technology 10, 779-783 (1992);Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368 812-13(1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995); as well as U.S. Pat. Nos.4,816,567; 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and,5,661,016). The antibodies or derivatives therefrom may also beconjugated to therapeutic moieties such as cytotoxic drugs or toxins, oractive fragments thereof such as diptheria A chain, exotoxin A chain,ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin,among others. Cytotoxic agents may also include radiochemicals.Antibodies and their derivatives may be incorporated into compositionsof the invention for use in vitro or in vivo.

[0063] Nucleic acids, proteins, or derivatives thereof representing animmunogenic target may be used in assays to determine the presence of adisease state in a patient, to predict prognosis, or to determine theeffectiveness of a chemotherapeutic or other treatment regimen.Expression profiles, performed as is known in the art, may be used todetermine the relative level of expression of the immunogenic target.The level of expression may then be correlated with base levels todetermine whether a particular disease is present within the patient,the patient's prognosis, or whether a particular treatment regimen iseffective. For example, if the patient is being treated with aparticular chemotherapeutic regimen, an decreased level of expression ofan immunogenic target in the patient's tissues (i.e., in peripheralblood) may indicate the regimen is decreasing the cancer load in thathost. Similarly, if the level of expresssion is increasing, anothertherapeutic modality may need to be utilized. In one embodiment, nucleicacid probes corresponding to a nucleic acid encoding an immunogenictarget may be attached to a biochip, as is known in the art, for thedetection and quantification of expression in the host.

[0064] It is also possible to use nucleic acids, proteins, derivativestherefrom, or antibodies thereto as reagents in drug screening assays.The reagents may be used to ascertain the effect of a drug candidate onthe expression of the immunogenic target in a cell line, or a cell ortissue of a patient. The expression profiling technique may be combinedwith high throughput screening techniques to allow rapid identificationof useful compounds and monitor the effectiveness of treatment with adrug candidate (see, for example, Zlokarnik, et al., Science 279, 84-8(1998)). Drug candidates may be chemical compounds, nucleic acids,proteins, antibodies, or derivatives therefrom, whether naturallyoccurring or synthetically derived. Drug candidates thus identified maybe utilized, among other uses, as pharmaceutical compositions foradministration to patients or for use in further screening assays.

[0065] Administration of a composition of the present invention to ahost may be accomplished using any of a variety of techniques known tothose of skill in the art. The composition(s) may be processed inaccordance with conventional methods of pharmacy to produce medicinalagents for administration to patients, including humans and othermammals (i.e., a “pharmaceutical composition”). The pharmaceuticalcomposition is preferably made in the form of a dosage unit containing agiven amount of DNA, viral vector particles, polypeptide or peptide, forexample. A suitable daily dose for a human or other mammal may varywidely depending on the condition of the patient and other factors, but,once again, can be determined using routine methods.

[0066] The pharmaceutical composition may be administered orally,parentally, by inhalation spray, rectally, intranodally, or topically indosage unit formulations containing conventional pharmaceuticallyacceptable carriers, adjuvants, and vehicles. The term “pharmaceuticallyacceptable carrier” or “physiologically acceptable carrier” as usedherein refers to one or more formulation materials suitable foraccomplishing or enhancing the delivery of a nucleic acid, polypeptide,or peptide as a pharmaceutical composition. A “pharmaceuticalcomposition” is a composition comprising a therapeutically effectiveamount of a nucleic acid or polypeptide. The terms “effective amount”and “therapeutically effective amount” each refer to the amount of anucleic acid or polypeptide used to induce or enhance an effectiveimmune response. It is preferred that compositions of the presentinvention provide for the induction or enhancement of an anti-tumorimmune response in a host which protects the host from the developmentof a tumor and/or allows the host to eliminate an existing tumor fromthe body.

[0067] For oral administration, the pharmaceutical composition may be ofany of several forms including, for example, a capsule, a tablet, asuspension, or liquid, among others. Liquids may be administered byinjection as a composition with suitable carriers including saline,dextrose, or water. The term parenteral as used herein includessubcutaneous, intravenous, intramuscular, intrasternal, infusion, orintraperitoneal administration. Suppositories for rectal administrationof the drug can be prepared by mixing the drug with a suitablenon-irritating excipient such as cocoa butter and polyethylene glycolsthat are solid at ordinary temperatures but liquid at the rectaltemperature.

[0068] The dosage regimen for immunizing a host or otherwise treating adisorder or a disease with a composition of this invention is based on avariety of factors, including the type of disease, the age, weight, sex,medical condition of the patient, the severity of the condition, theroute of administration, and the particular compound employed. Forexample, a poxviral vector may be administered as a compositioncomprising 1×10⁶ infectious particles per dose. Thus, the dosage regimenmay vary widely, but can be determined routinely using standard methods.

[0069] A prime-boost regimen may also be utilized (WO 01/30382 A1) inwhich the targeted immunogen is initially administered in a priming stepin one form followed by a boosting step in which the targeted immunogenis administered in another form. The form of the targeted immunogen inthe priming and boosting steps are different. For instance, if thepriming step utilized a nucleic acid, the boost may be administered as apeptide. Similarly, where a priming step utilized one type ofrecombinant virus (i.e., ALVAC), the boost step may utilize another typeof virus (i.e., NYVAC). This prime-boost method of administration hasbeen shown to induce strong immunological responses.

[0070] While the compositions of the invention can be administered asthe sole active pharmaceutical agent, they can also be used incombination with one or more other compositions or agents (i.e., otherimmunogenic targets, co-stimulatory molecules, adjuvants). Whenadministered as a combination, the individual components can beformulated as separate compositions administered at the same time ordifferent times, or the components can be combined as a singlecomposition.

[0071] Injectable preparations, such as sterile injectable aqueous oroleaginous suspensions, may be formulated according to known methodsusing suitable dispersing or wetting agents and suspending agents. Theinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent.Suitable vehicles and solvents that may be employed are water, Ringer'ssolution, and isotonic sodium chloride solution, among others. Forinstance, a viral vector such as a poxvirus may be prepared in 0.4%NaCl. In addition, sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose, any bland fixed oil maybe employed, including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

[0072] For topical administration, a suitable topical dose of acomposition may be administered one to four, and preferably two or threetimes daily. The dose may also be administered with intervening daysduring which no does is applied. Suitable compositions may comprise from0.001% to 10% w/w, for example, from 1% to 2% by weight of theformulation, although it may comprise as much as 10% w/w, but preferablynot more than 5% w/w, and more preferably from 0.1% to 1% of theformulation. Formulations suitable for topical administration includeliquid or semi-liquid preparations suitable for penetration through theskin (e.g., liniments, lotions, ointments, creams, or pastes) and dropssuitable for administration to the eye, ear, or nose.

[0073] The pharmaceutical compositions may also be prepared in a solidform (including granules, powders or suppositories). The pharmaceuticalcompositions may be subjected to conventional pharmaceutical operationssuch as sterilization and/or may contain conventional adjuvants, such aspreservatives, stabilizers, wetting agents, emulsifiers, buffers etc.Solid dosage forms for oral administration may include capsules,tablets, pills, powders, and granules. In such solid dosage forms, theactive compound may be admixed with at least one inert diluent such assucrose, lactose, or starch. Such dosage forms may also comprise, as innormal practice, additional substances other than inert diluents, e.g.,lubricating agents such as magnesium stearate. In the case of capsules,tablets, and pills, the dosage forms may also comprise buffering agents.Tablets and pills can additionally be prepared with enteric coatings.Liquid dosage forms for oral administration may include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions may also comprise adjuvants, such as wetting sweetening,flavoring, and perfuming agents.

[0074] Pharmaceutical compositions comprising a nucleic acid orpolypeptide of the present invention may take any of several forms andmay be administered by any of several routes. In preferred embodiments,the compositions are administered via a parenteral route (intradermal,intramuscular or subcutaneous) to induce an immune response in the host.Alternatively, the composition may be administered directly into a lymphnode (intranodal) or tumor mass (i.e., intratumoral administration). Forexample, the dose could be administered subcutaneously at days 0, 7, and14. Suitable methods for immunization using compositions comprising TAsare known in the art, as shown for p53 (Hollstein et al., 1991), p21-ras(Almoguera et al., 1988), HER-2 (Fendly et al., 1990), themelanoma-associated antigens (MAGE-1; MAGE-2) (van der Bruggen et al.,1991), p97 (Hu et al., 1988), melanoma-associated antigen E (WO99/30737) and carcinoembryonic antigen (CEA) (Kantor et al., 1993;Fishbein et al., 1992; Kaufman et al., 1991), among others.

[0075] Preferred embodiments of administratable compositions include,for example, nucleic acids or polypeptides in liquid preparations suchas suspensions, syrups, or elixirs. Preferred injectable preparationsinclude, for example, nucleic acids or polypeptides suitable forparental, subcutaneous, intradermal, intramuscular or intravenousadministration such as sterile suspensions or emulsions. For example, arecombinant poxvirus may be in admixture with a suitable carrier,diluent, or excipient such as sterile water, physiological saline,glucose or the like. The composition may also be provided in lyophilizedform for reconstituting, for instance, in isotonic aqueous, salinebuffer. In addition, the compositions can be co-administered orsequentially administered with other antineoplastic, anti-tumor oranti-cancer agents and/or with agents which reduce or alleviate illeffects of antineoplastic, anti-tumor or anti-cancer agents.

[0076] A kit comprising a composition of the present invention is alsoprovided. The kit can include a separate container containing a suitablecarrier, diluent or excipient. The kit can also include an additionalanti-cancer, anti-tumor or antineoplastic agent and/or an agent thatreduces or alleviates ill effects of antineoplastic, anti-tumor oranti-cancer agents for co- or sequential-administration. Additionally,the kit can include instructions for mixing or combining ingredientsand/or administration.

[0077] A better understanding of the present invention and of its manyadvantages will be had from the following examples, given by way ofillustration.

EXAMPLES Example 1 BFA4 Tumor Antigen

[0078] The BFA4 sequence was found to be the “trichorhinophalangealsyndrome 1” (TRPS-1) gene (Genebank ID #6684533; Momeniet et al, NatureGenetics, 24(1), 71-74,2000), a known transcription factor with nofunction attributed previously in any form of cancer. The BFA4 cDNAsequence is shown in FIG. 1 and the deduced amino acid sequence is shownin FIG. 2.

[0079] A. BFA4 Peptides and Polyclonal Antisera

[0080] For monitoring purposes, rabbit anti-BFA4 polyclonal antibodieswere generated. Six peptides (22-mers) were designed and synthesized toelicit antibody response to BFA4, as shown below: CLP 2589MVRKKNPPLRNVASEGEGQILE BFA4 (1-22) CLP 2590 SPKATEETGQAQSGQANCQGLS BFA4(157-178) CLP 2591 VAKPSEKNSNKSIPALQSSDSG BFA4 (371-392) CLP 2592NHLQGSDGQQSVKESKEHSCTK BFA4 (649-670) CLP 2593 NGEQIIRRRTRKRLNPEALQAEBFA4 (940-961) CLP 2594 ANGASKEKTKAPPNVKNEGPLNV BFA4 (1178-1199)

[0081] Rabbits were immunized with the peptides, serum was isolated, andthe following antibody titers were observed: Rabbit # Peptide Titer(Bleed 2) Titer (Final Bleed) 1, 2 CLP2589 800000, 1600000 2560000,2560000 3, 4 CLP2590 12800, 6400 40000, 40000 5, 6 CLP2591 400000,400000 320000, 320000 7, 8 CLP2592 25600, 12800 80000, 40000 9, 10CLP2593 3200000, 51200 2560000, 160000 11, 12 CLP2594 409600, 409600320000, 320000

[0082] These peptides were also modified by coupling with KLH peptidesto enhance immune responses as shown below: BFA4 (1-22)KLH-MVRKKNPPLRNVASEGEG (CLP-2589) QILE BFA4 (157-178)KLH-SPKATEETGQAQSGQANC (CLP-2590) QGLS BFA4 (371-392)KLH-VAKPSEKNSNKSIPALQS (CLP-2591) SDSG BFA4 (649-670)KLH-NHLQGSDGQQSVKESKEH (CLP-2592) SCTK BFA4 (940-961)KLH-NGEQIIRRRTRKRLNPEA (CLP-2593) LQAE BFA4 (1178-1200)KLH-ANGASKEKTKAPPNVKNE (CLP-2594) GPLNV

[0083] The pcDNA3.2BFA4 (3.6 mg) was also used for DNA immunization togenerate polyclonal sera in chickens.

[0084] B. Cloning of BFA4

[0085] Complete cDNA sequence for BFA4 is ˜10 kb and gene is expressedin BT474 ductal carcinoma cells. Primers 7717 (forward primer) and 7723(reverse primer) were designed to amplify full-length BFA4 gene byamplification of 4 kb, 7 kb or 10 kb products by RT-PCR.

[0086] Primer 7717: BFA4-BamH1/F1 (5′ end forward) with Kozak:

[0087] 5′ CGGGATCCACCATGGTCCGGAAAAAGAACCCC 3′(BamHI for DNA3.1, MP76)

[0088] Primer 7723: BFA4-BamHI/R1 (3′ end reverse 4 kb):

[0089] 5′ CGGGATCCCTCTTTAGGTTTTCCATTTTTTTCCAC 3′ (BamHI for DNA3.1,MP76)

[0090] Ten mg of total RNA isolated and frozen in different batches fromBT-474 cells using Trizol as indicated by the manufacturer (Gibco BRL)was used in RT-PCR to amplify the BFA4 gene. RT-PCR conditions wereoptimized using Taq Platinum High Fidelity enzyme, OPC (OligoPurification Cartridge; Applied Biosystems) purified primers andpurified total RNA/polyA mRNA (BT 474 cells). Optimization resulted in a4.0 kb fragment as a single band.

[0091] To re-amplify the BFA4 sequence, mRNA was treated with DNase permanufacturers' instructions (Gibco BRL). The 4 kb DNA was reamplifiedusing PCR using primers 7717 and 7723 primers (10 pmole/microlitre) andTaq Platinum High Fidelity polymerase (GIBCO BRL) enzyme. Thermocyclerconditions for both sets of reactions were as under: 94° C. (2 min),followed by 30 cycles of 94° C. (30 sec), 52° C. (30 sec), 67° C. (4min) and 67° C. (5 min) and finally 40° C. for 10 min. Three BFA4 cloneswere identified after pCR2.1/TOPO-TA cloning.

[0092] Several mutations were identified during analysis of the BFA4sequence. To correct these sequences, the BamHI/XhoI fragment (5′) ofthe BFA4 gene from clone JB-3552-1-2 (pCR2.1/TOPO/BFA4) was exchangedwith the XhoI/BamHI fragment (3′) of the BFA4 gene from cloneJB-3552-1-4 (pCR2.1/TOPO/BFA4). This recombined fragment was thenligated into pMCS5 BamHI/CAP. Clone JB-3624-1-5 was generated and foundto contain the correct sequence.

[0093] Nucleotide 344 of the isolated BFA4 clone was different from thereported sequence (C in BFA4, T in TRPS-1). The change resulted in a pheto ser amino acid change. To change this sequence to the reportedsequence, the EcoRI/BglII fragment (5′) of the BFA4 gene from cloneJB-3552-1-2 (pCR2.1/TOPO/BFA4) was subcloned into pUC8:2 to generateclone JB-3631-2. This clone was used as a template for Quickchange(Stratagene) mutagenesis to change amino acid 115 of the BFA4 proteinfrom a serine to a phenylalanine as in the TRPS1 protein. The selectedclone was JB-3648-2-3. Mutagenesis was also repeated with pMCS5 BFA4(BT474) as a template for Quickchange (Stratagene) mutagenesis to changeamino acid 115 of the BFA4 protein from a serine to a phenylalanine asin the TRPSI protein. Several clones were found to be correct by DNAsequencing and one of the clones (JB-3685-1-18) was used for furthersubcloning.

[0094] JB-3685-1-18 was then used to subclone the BFA4 coding sequenceinto the BamHI sites of four different expression vectors: 1) thepoxviral (NYVAC) vector pSD554VC (COPAK/H6; JB-3707-1-7); 2)pcDNA3.1/Zeo (+) (JB-3707-3-2); 3) pCAMycHis (JB-3707-5-1); and, 4)Semiliki Forest virus alphaviral replicon vector pMP76 (JB-3735-1-23).The BFA4 coding sequence within JB-3707-1-7, JB-3707-5-1, andJB-3735-1-23 was confirmed by DNA sequencing.

[0095] A stop codon was introduced near the end of the cloned sequencein the pcDNA3.1/Zeo/BFA4 construct (JB-3707-3-2). A unique EcoRl sitewas opened and filled in to introduce a stop codon in-frame with BFA4coding sequence. Several putative clones were identified by the loss ofEcoRl site, however three clones (JB-3756-1-2; JB-3756-3-1; andJB-3756-4-1) were sequenced. All three were found to be correct in thearea of the fill-in. Clone JB-3756-3-1 identified as having the correctsequence and orientation.

[0096] Myc and myc/his tags (Evans et al, 1985) were introduced usingoligonucleotides, which were annealed and ligated into thepcDNA3.1/Zeo/BFA4 construct (JB-3707-3-2) at the EcoRI/EcoRV sites.Several clones were obtained for these constructs. Three clones havingthe correct sequences and orientations were obtained: 1)PcDNA3.1/Zeo/BFA4/myc-tag (JB-3773-1-2); 2) PcDNA3.1/Zeo/BFA4/mychis-tag(JB-3773-2-1); and, 3) PcDNA3.1/Zeo/BFA4/mychis-tag (JB-3773-2-2).

[0097] C. Expression of BFA4

[0098] 1. Expression From Poxviral Vectors

[0099] The pSD554VC (COPAK/H6; JB-3707-1-7) vector was used to generateNYVAC-BFA4 virus. In vitro recombination was performed with plasmidCOPAK/H6/BFA4 and NYVAC in RK13/CEF cells. NYVAC-BFA4(vP2033-NYVAC-RK13) was generated and amplified to P3 level aftercompletion of three enrichments with final stock concentrations of1.12×10⁹/ml (10 ml). Vero cells were infected with NYVAC-BFA4 at anM.O.I. of 0.5 pfu/cell. Lysates and media were harvested 24 hpost-infection to confirm expression of BFA4 protein. One-twentieth ofthe concentrated media and {fraction (1/40)} of the lysate were loadedonto a western blot and incubated with rabbit antisera against the BFA4peptides CLP 2589, 2591, 2598 and 2594 (see above for peptide sequencesand preparation of anti-BFA4 antisera). An approximate 120 kD band wasdetected in both the lysate and the concentrated media ofNYVAC-BFA4-infected Vero cells which was not evident in either Verocontrol cells (“mock-infected”), Vero cells infected with the parentalNYVAC virus, or concentrated media.

[0100] 2. Expression From pcDNA3.1-based Vectors

[0101] Transient transfection studies were performed to verifyexpression of BFA4 from the pcDNA-based vectors and to analyze qualityof polyclonal sera raised against BFA4 peptides. The followingconstructs were used to study expression of BFA4 gene: pcDNA 3.1zeoR/BFA4, pMP76/BFA4, pcDNA 3.1 zeor/BFA4/Myc tag and pcDNA 3.1zeoR/BFA4/MycHis tag. BFA4 expression plasmids (5 μg and 10 μg) wereco-transfected with pGL3 Luciferase (1 □g) (Promega) with the Geneporter reagent (Gene Therapy Systems) as the transfection reagent. At 48h post-transfection, whole cell extract was prepared by scraping cellsin cell lysis reagent (200 μl) and 1 cycle of freeze-thaw (−20° C.freeze, 37° C. thaw). Transfection efficiency was quantitated byanalyzing expression of the luciferase reporter gene by measuringRelative Luciferase Units (RLU) in duplicate. Similar RLU values wereobtained in the samples co-transfected with luciferase construct in thepresence and absence of BFA4 expression vectors. There was nosignificant difference observed in toxicity or RLU values withdifferential amount (5 μg and 10 μg) of BFA4 expression vectors.Preliminary western blot analysis using alkaline phosphatase system withthe CHOKI cell extracts (pCDNA3.1 /zeo/BFA4/MycHisTag) and an anti-BFA4polyclonal antisera, revealed a band at approximately 120 kDa band inextracts of BFA4 vector-transfected cells.

[0102] A stable transfection study was initiated to obtain stable clonesof BFA4 expressing COS A2 cells. These cells are useful for in vitrostimulation assays. pcDNA 3.1 zeo^(R)/BFA4 (2.5 μg and 20 μg), and pcDNA3.1 zeo^(R)/BFA4/MycHis tag (2.5 μg) were used to study expression ofBFA4). pGL3 Luciferase (2.5 μg) was used as a control vector to monitortransfection efficiency. The Gene porter reagent was used to facilitatetransfection of DNA vectors. After 48 h post-transfection, whole cellextract were prepared by scraping cells in the cell lysis reagent (200μl) and 1 cycle of freeze-thaw at −20° C./37° C. for first experiment.Transfected cells obtained from the second experiment were trypsinized,frozen stock established and cells were plated in increasingconcentrations of Zeocin (0, 250, 500, 750 and 1000 μg/ml).Non-transfected CosA2cells survived at 60-80% confluency for three weeksat 100 μg/ml (Zeocin) and 10% confluency at 250 μg/ml (Zeocin). However,after three weeks, at higher drug concentration (500-1000 μg/ml), livecells were not observed in the plates containing non-transfected cellsand high Zeocin concentration (500-1000 μg/ml).

[0103] Several Zeocin-resistant clones growing in differential drugconcentrations (Zeocin-250, 500, 750 and 1000 μg/ml) were picked from 10cm plates after three weeks. These clones were further expanded in a 3.5cm plate(s) in the presence of Zeocin at 500, 750 and 1000 μg/ml. Frozenlots of these clones were prepared and several clones from each pool(pcDNA 3.1 zeo^(R)/BFA4, and pcDNA 3.1 zeor/BFA4/MycHis tag) wereexpanded to T75 cm² flasks in the presence of Zeocin at 1 mg/ml. Fiveclones from each pool (pcDNA 3.1 zeo^(R)/BFA4, and pcDNA 3.1zeo^(R)/BFA4/MycHis tag) were expanded to T75 cm² flasks in the presenceof Zeocin at 1 mg/ml. Cells are maintained under Zeocin drug (1 mg/ml)selection. Six clones were used in BFA4 peptide-pulsed targetexperiment, and two clones were found to express BFA4 at a moderatelevel by immunological assays. The non-adherent cell lines K562A2 andEL4A2 were also transfected with these vectors to generate stable celllines.

[0104] 3. Prokaryotic Expression Vector

[0105] The BamHI-Xho-1 fragment (1.5 Kbp) fragment encoding N-terminal54kDa BFDA4 from pCDNA3.1/BFA4 was cloned into pGEX4T1-6His (Veritas)plasmid. This vector contains the tac promoter followed by theN-terminal glutathione S-transferase (GST˜26 kDa) and a hexahistidinetag to C terminus of the GST fusion protein.

[0106] The BFA4-N54 expression plasmid was transformed into BL21 cellsand grown at 25° C. in antibiotic selection medium (2 L culture) to anOD (600 nm) and thereafter induced with 1 mM IPTG. GST-BFA4-N54 wasfound to be soluble protein. Clarified extract of the soluble fractionwas adsorbed batchwise to glutathione-Sepharose 4B and eluted with 10 mMreduced glutathione. Fractions were analyzed after estimation of proteinconcentration and TCA precipitation. Specific polypeptide of Mr=85 kDain the eluate was confirmed by SDS-PAGE. The recombinant protein waspurified by gluathione-Sepharose was absorbed on a NiNTA column forfurther purification. The bound protein was eluted with 0.25M imidazole.The protein was dialyzed versus TBS containing 40% Glycerol, resultingin 4.5 mg GST-BFA4-N54-6 His (N terminus BFA4 protein) protein.Expression of BFA4 was confirmed using the rabbit anti-BFA4 polyclonalantibody by western blot.

[0107] D. Therapeutic Anti-BFA4 Immune Responses

[0108] 1. BFA4 Peptides

[0109] In addition to genetic immunization vectors for BFA4,immunological reagents for BFA4 have been generated. A library of 100nonamer peptides spanning the BFA4 gene product was synthesized. Thepeptides were chosen based on their potential ability to bind toHLA-A*0201. Table V lists 100 nonamer peptide epitopes for HLA-A*0201from the BFA4 protein tested (see below): PEPTIDE POSITION DESIGNATIONSEQUENCE IN PROTEIN CLP-2421 MVRKKNPPL BFA4 (1-9)i″ CLP-2422 KKNPPLRNVBFA4 (4-12)i″ CLP-2423 VASEGEGQI BFA4 (12-20)i″ CLP-2424 QILEPIGTE BFA4(19-27)i″ CLP-2425 RNMLAFSFP BFA4 (108-116)i″ CLP-2426 NMLAFSFPA BFA4(109-117)i″ CLP-2427 MLAFSFPAA BFA4 (110-118)i″ CLP-2428 FSFPAAGGV BFA4(113-121)i″ CLP-2429 AAGGVCEPL BFA4 (117-125)i″ CLP-2430 SGQANCQGL BFA4(170-178)i″ CLP-2431 ANCQGLSPV BFA4 (172-180)i″ CLP-2432 GLSPVSVAS BFA4(176-184)i″ CLP-2433 SVASKNPQV BFA4 (181-189)i″ CLP-2434 RLNKSKTDL BFA4(196-204)i″ CLP-2435 NDNPDPAPL BFA4 (207-215)i″ CLP-2436 DPAPLSPEL BFA4(211-219)i″ CLP-2437 ELQDFKCNI BFA4 (218-216)i″ CLP-2438 GLHNRTRQD BFA4(249-257)i″ CLP-2439 ELDSKILAL BFA4 (259-267)i″ CLP-2440 KILALHNMV BFA4(263-271)i″ CLP-2441 ALHNMVQFS BFA4 (266-284)i″ CLP-2442 VNRSVFSGV BFA4(282-290)i″ CLP-2443 FSGVLQDIN BFA4 (287-295)i″ CLP-2444 DINSSRPVL BFA4(293-301)i″ CLP-2445 VLLNGTYDV BFA4 (300-308)i″ CLP-2446 FCNFTYMGN BFA4(337-345)i″ CLP-2447 YMGNSSTEL BFA4 (342-350)i″ CLP-2448 FLQTHPNKI BFA4(354-362)i″ CLP-2449 KASLPSSEV BFA4 (363-371)i″ CLP-2450 DLGKWQDKI BFA4(393-401)i″ CLP-2451 VKAGDDTPV BFA4 (403-411)i″ CLP-2452 FSCESSSSL BFA4(441-449)i″ CLP-2453 KLLEHYGKQ BFA4 (450-458)i″ CLP-2454 GLNPELNDK BFA4(466-474)i″ CLP-2455 GSVINQNDL BFA4 (478-486)i″ CLP-2456 SVINQNDLA BFA4(479-487)i″ CLP-2457 FCDFRYSKS BFA4 (527-535)i″ CLP-2458 SHGPDVIVV BFA4(535-543)i″ CLP-2459 PLLRHYQQL BFA4 (545-553)i″ CLP-2460 GLCSPEKHL BFA4(570-578)i″ CLP-2461 HLGEITYPF BFA4 (577-585)i″ CLP-2462 LGEITYPFA BFA4(578-586)i″ CLP-2463 HCALLLLHL BFA4 (594-602)i″ CLP-2464 ALLLLHLSP BFA4(596-604)i″ CLP-2465 LLLLHLSPG BFA4 (597-605)i″ CLP-2466 LLLHLSPGA BFA4(598-606)i″ CLP-2467 LLHLSPGAA BFA4 (599-607)i″ CLP-2468 FTTPDVDVL BFA4(621-629)i″ CLP-2469 TTPDVDVLL BFA4 (622-630)i″ CLP-2470 VLLFHYESV BFA4(628-636)i″ CLP-2471 FITQVEEEI BFA4 (673-681)i″ CLP-2472 FTAADTQSL BFA4(699-707)i″ CLP-2473 SLLEHFNTV BFA4 (706-714)i″ CLP-2474 STIKEEPKI BFA4(734-742)i″ CLP-2475 KIDFRVYNL BFA4 (741-749)i″ CLP-2476 NLLTPDSKM BFA4(748-756)i″ CLP-2479 VTWRGADIL BFA4 (792-800)i″ CLP-2480 ILRGSPSYT BFA4(799-807)i″ CLP-2481 YTQASLGLL BFA4 (806-814)i″ CLP-2482 ASLGLLTPV BFA4(809-817)i″ CLP-2483 GLLTPVSGT BFA4 (812-820)i″ CLP-2484 GTQEQTKTL BFA4(819-827)i″ CLP-2485 KTLRDSPNV BFA4 (825-833)i″ CLP-2486 HLARPIYGL BFA4(837-845)i″ CLP-2487 PIYGLAVET BFA4 (841-849)i″ CLP-2488 LAVETKGFL BFA4(845-853)i″ CLP-2489 FLQGAPAGG BFA4 (852-860)i″ CLP-2490 AGGEKSGAL BFA4(858-866)i″ CLP-2491 GALPQQYPA BFA4 (864-872)i″ CLP-2492 ALPQQYPAS BFA4(865-873)i″ CLP-2493 FCANCLTTK BFA4 (895-903)i″ CLP-2494 ANGGYVCNA BFA4(911-919)i″ CLP-2495 NACGLYQKL BFA4 (918-926)i″ CLP-2496 GLYQKLHST BFA4(921-929)i″ CLP-2497 KLHSTPRPL BFA4 (925-933)i″ CLP-2498 STPRPLNII BFA4(928-936)i″ CLP-2499 RLNPEALQA BFA4 (952-960)i″ CLP-2500 VLVSQTLDI BFA4(1020-1028)i″ CLP-2501 DIHKRMQPL BFA4 (1027-1035)i″ CLP-2502 RMQPLHIQIBFA4 (1031-1039)i″ CLP-2503 YPLFGLPFV BFA4 (1092-1100)i″ CLP-2504GLPFVHNDF BFA4 (1096-1104)i″ CLP-2505 FVHNDFQSE BFA4 (1099-1107)i″CLP-2506 SVPGNPHYL BFA4 (1120-1128)i″ CLP-2507 GNPHYLSHV BFA4(1123-1131)i″ CLP-2508 HYLSHVPGL BFA4 (1126-1134)i″ CLP-2509 YVPYPTFNLBFA4 (1141-1149)i″ CLP-2510 FNLPPHFSA BFA4 (1147-1155)i″ CLP-2511NLPPHFSAV BFA4 (1148-1156)i″ CLP-2512 SAVGSDNDI BFA4 (1154-1162)i″CLP-2513 KNEGPLNVV BFA4 (1192-1200)i″ CLP-2514 TKCVHCGIV BFA4(1215-1223)i″ CLP-2515 CVHCGIVFL BFA4 (1217-1225)i″ CLP-2516 CGIVFLDEVBFA4 (1220-1228)i″ CLP-2517 FLDEVMYAL BFA4 (1224-1232)i″ CLP-2518VMYALHMSC BFA4 (1228-1236)i″ CLP-2519 FQCSICQHL BFA4 (1243-1251)i″CLP-2520 GLHRNNAQV BFA4 (1265-1273)i″

[0110] The peptide library was pooled into separate groups containing7-10 different peptides for immunological testing as shown in Table VI(see below). In addition to a peptide library spanning BFA4, arecombinant protein spanning the N-terminal 300 amino acids (positions1-300) has been synthesized and purified from E. coli. PEPTIDE PEPTIDEGROUP NUMBER SEQUENCE 1 CLP-2421 MVRKKNPPL CLP-2422 KKNPPLRNV CLP-2423VASEGEGQI CLP-2424 QILEPIGTE CLP-2425 RNMLAFSFP CLP-2426 NMLAFSFPACLP-2427 MLAFSFPAA CLP-2428 FSFPAAGGV CLP-2429 AAGGVCEPL CLP-2430SGQANCQGL 2 CLP-2431 ANCQGLSPV CLP-2432 GLSPVSVAS CLP-2433 SVASKNPQVCLP-2434 RLNKSKTDL CLP-2435 NDNPDPAPL CLP-2436 DPAPLSPEL CLP-2437ELQDFKCNI CLP-2438 GLHNRTRQD CLP-2439 ELDSKILAL CLP-2440 KILALHNMV 3CLP-2441 ALHNMVQFS CLP-2442 VNRSVFSGV CLP-2443 FSGVLQDIN CLP-2444DINSSRPVL CLP-2445 VLLNGTYDV CLP-2446 FCNFTYMGN CLP-2447 YMGNSSTELCLP-2448 FLQTHPNKI CLP-2449 KASLPSSEV CLP-2450 DLGKWQDKI 4 CLP-2451VKAGDDTPV CLP-2452 FSCESSSSL CLP-2453 KLLEHYGKQ CLP-2454 GLNPELNDKCLP-2455 GSVINQNDL CLP-2456 SVINQNDLA CLP-2457 FCDFRYSKS CLP-2458SHGPDVIVV CLP-2459 PLLRHYQQL CLP-2460 GLCSPEKHL 5 CLP-2461 HLGEITYPFCLP-2462 LGEITYPFA CLP-2463 HCALLLLHL CLP-2464 ALLLLHLSP CLP-2465LLLLHLSPG CLP-2466 LLLHLSPGA CLP-2467 LLHLSPGAA CLP-2468 FTTPDVDVLCLP-2469 TTPDVDVLL CLP-2470 VLLFHYESV 6 CLP-2471 FITQVEEEI CLP-2472FTAADTQSL CLP-2473 SLLEHFNTV CLP-2474 STIKEEPKI CLP-2475 KIDFRVYNLCLP-2476 NLLTPDSKM CLP-2477 KMGEPVSES CLP-2478 GLKEKVWTE CLP-2479VTWRGADIL CLP-2480 ILRGSPSYT 7 CLP-2481 YTQASLGLL CLP-2482 ASLGLLTPVCLP-2483 GLLTPVSGT CLP-2484 GTQEQTKTL CLP-2485 KTLRDSPNV CLP-2486HLARPIYGL CLP-2487 PIYGLAVET CLP-2488 LAVETKGFL CLP-2489 FLQGAPAGGCLP-2490 AGGEKSGAL 8 CLP-2491 GALPQQYPA CLP-2492 ALPQQYPAS CLP-2493FCANCLTTK CLP-2494 ANGGYVCNA CLP-2495 NACGLYQKL CLP-2496 GLYQKLHSTCLP-2497 KLHSTPRPL CLP-2498 STPRPLNII CLP-2499 RLNPEALQA CLP-2500VLVSQTLDI 9 CLP-2501 DIHKRMQPL CLP-2502 RMQPLHIQI CLP-2503 YPLFGLPFVCLP-2504 GLPFVHNDF CLP-2505 FVHNDFQSE CLP-2506 SVPGNPHYL CLP-2507GNPHYLSHV CLP-2508 HYLSHVPGL CLP-2509 YVPYPTFNL CLP-2510 FNLPPHFSA 10CLP-2511 NLPPHFSAV CLP-2512 SAVGSDNDI CLP-2513 KNEGPLNVV CLP-2514TKCVHCGIV CLP-2515 CVHCGIVFL CLP-2516 CGIVFLDEV CLP-2517 FLDEVMYALCLP-2518 VMYALHMSC CLP-2519 FQCSICQHL CLP-2520 GLHRNNAQV

[0111] 2. Immune Reactivity of BFA4 Peptides and Generation of HumanEffector T cells:

[0112] The BFA4 peptides were grouped into different pools of 7-10peptides for immunological testing. Dissolved peptide pools were pulsedonto autologous HLA-A*0201 dendritic cells and used to activateautologous T-cell-enriched PBMC preparations. Activated T cells fromeach peptide-pool-stimulated culture were re-stimulated another 3 to 5times using CD40L-activated autologous B-cells. IFN-γ ELISPOT analysisand assays for CTL killing of peptide-pulsed target cells was performedto demonstrate the immunogenicity of these epitopes from BFA4.

[0113] Human T cells demonstrated effector cell activity against anumber of pools of peptides from the BFA4 protein, as shown by theirability to secrete IFN-γ in ELISPOT assays. These experiments wererepeated after different rounds of APC stimulation resulting in the samereactive peptide groups. Peptide groups 1, 2, 4, 5, 6, 7, 8, 9, and 10were found to be immunoreactive in these assays. Subsequently, thesereactive peptide groups were de-convoluted in additional IFN-γ ELISPOTassays in which single peptides from each group were tested separately.The individual peptides from BFA4 peptide groups 1, 5 6, 7, 8, 9, and 10in ELISPOT assays. This analysis revealed a number of individualstrongly reactive peptides from the BFA4 protein recognized by human Tcells. It was also observed that many of these single peptides alsoinduced CTL activity killing peptide-loaded human T2 lymphoma celltargets. These peptides are listed in Table VII: TABLE VII List ofhighly immunoreactive peptides from BFA4 Strong IFN-γ Killing Strong CTLKilling CLP 2425 RNMLAFSFP CLP 2425 RNMLAFSFP CLP 2426 NMLAFSFPA CLP2426 NMLAFSFPA CLP 2427 MLAFSFPAA CLP 2427 MLAFSFPAA CLP 2461 HLGEITYPFCLP 2468 FTTPDVDVL CLP 2468 FTTPDVDVL CLP 2470 VLLFHYESV CLP 2470VLLFHYYESV CLP 2474 KIDFRVYNL CLP 2482 ASLGLLTPV CLP 2482 ASLGLLTPV CLP2486 HLARPIYGL CLP 2486 HLARPIYGL CLP 2495 NACGLYQKL CLP 2495 NACGLYQKLCLP 2497 KLHSTPRPL CLP 2499 RLNPEALQA CLP 2499 RLNPEALQA CLP 2503YPLFGLPFV CLP 2509 YVPYPTFNL CLP 2509 YVPYPTFNL CLP 2511 NLPPHFSAV CLP2518 VMYALHMSC CLP 2520 GLHRNNAQV CLP 2520 GLHRNNAQV

[0114] D. Immune Responses Against BFA4 After Immunization In Vivo:

[0115] The pcDNA3.1/Zeo-BFA4 plasmid was used to immunize transgenicmice expressing a hybrid HLA-A*0201 α1α2 domain fused to a murine Kb α3domain in C57BL/6 mice (A2-Kb mice). IFN-γ ELISPOT analysis using thegroups of pooled peptides after DNA immunization and removal ofactivated spleen cells revealed a number of reactive BFA4 peptidegroups. Some of these groups (especially group 7 and 8) also reactedstrongly in human T-cell cultures suggesting that overlapping groups ofpeptides are recognized by human T cells and are naturally processed andpresented on HLA-A2 after vaccination.

[0116] Vaccination experiments were also performed with the NYVAC-BFA4and the MP76-18-BFA4 vectors in A2-Kb mice. Mice were immunizedsubcutaneously with 10-20 μg of MP-76-18-BFA4 and 1-2×10⁷ pfu vP2033(NYVAC-BFA4) and boosted 28 days later with the same amounts of eachvector. Re-stimulation of spleen cells from the immunized mice with thepools of BFA4 peptides revealed induction of IFN-γ production inresponse to BFA4 peptide groups 2, 3, 4, 5, 7, 9, and 10 in ELISPOTassays. Thus, the BFA4 gene encoded in a CMV promoter driven eukaryoticplasmid, NYVAC, or a Semliki replicase-based DNA plasmid, were allcapable of inducing T-cell responses against the BFA4 protein in vivo.

Example 2 BCY1 Tumor Antigen

[0117] The BCY1 gene was detected as a partial open reading frame (ORF)homologous to a nematode gene called “posterior-expressed maternalgene-3” (PEM-3) playing a role in posterior to anterior patterning inCaenorhabtidis elegans embryos. No previous involvement of this gene incancer has been documented.

[0118] A. BCY1 and Amino Acid DNA Sequences

[0119] A partial DNA sequence was originally determined for BCY1.Primers, 9616SXC and 9617SXC, are derived from the BCY I partial DNAsequence and are designed to clone BCY I by RT-PCR from Calu 6 totalRNA. The primers were designed such that the PCR product has BamHI sitesat both ends and an ATG start codon and a Kozak sequence at the 5′ end,as shown below: 9616SXC: 5′ CAGTACGGATCCACCATGGCCGAGCTGCGCCTGA AGGGC 3′9617SXC: 5′ CCACGAGGATCCTTAGGAGAATATTCGGATGGC TTGCG 3′

[0120] The 1.2 Kb expected amplicon was obtained using ThermoScriptRT-PCR System (Invitrogen) under optimized conditions. The PCR productsfrom three separate RT-PCR's were digested with BamHI and respectivelyinserted in pcDNA3. 1/Zeo(+). The resulting clones were MC50A6, MC50A8and MC50A19 from the first RT-PCR; MC54.21 from the second RT-PCR andMC55.29; and, MC55.32 from the third RT-PCR (FIG. 3). The followingprimers were utilized in sequencing the clones: 9620MC:5′ TAATACGACTCACTATAGGG 3′ 9621MC: 5′ TAGAAGGCACAGTCGAGG 3′ 9618MC:5′ GAAAACGACTTCCTGGCGGGGAG 3′ 9619MC: 5′ GCTCACCCAGGCGTGGGGCCTC 3′

[0121] DNA sequencing of all six clones indicated a consensus sequence,as shown in FIGS. 3A and B, having the following differences from theoriginal partial BCY1 sequence: a C to G substitution at position 1031resulting in an amino acid change of Ala to Gly; a GC deletion atposition 1032-1034 resulting in a Thr deletion; and, an A to Gsubstitution at position 1177 resulting in an amino acid change of Thrto Ala. Clones MC50A8 and MC55.29 are identical to the consensussequence. The amino acid sequence of BCY1 is shown in FIG. 3B.

[0122] B. Immunological Reagents for BCY1 Breast Cancer Antigen:

[0123] A library of 100 nonamer peptides spanning the BCY1 gene productwas synthesized. The peptides were chosen based on their potentialability to bind to HLA-A*0201. Table VIII lists 100 nonamer peptideepitopes for HLA-A*0201 from the BCY1 protein tested (see below): TABLEVIII Peptide Position Designation Sequence in Protein *CLP-2599VPVPTSEHV   2 *CLP-2602 PTSEHVAEI   5 *CLP-2609 EIVGRQCKI  12 *CLP-2616KIKALRAKT  19 *CLP-2618 KALRAKTNT  21 *CLP-2619 ALRAKTNTY  22 *CLP-2620LRAKTNTYI  23 *CLP-2624 TNTYIKTPV  27 *CLP-2627 YIKTPVRGE  30 *CLP-2630TPVRGEEPV  33 *CLP-2633 RGEEPVFMV  36 *CLP-2640 MVTGRREDV  43  CLP-2641VTGRREDVA  44 *CLP-2643 GRREDVATA  46  CLP-2647 DVATARREI  50  CLP-2648VATARREII  51 *CLP-2650 TARREIISA  53 *CLP-2651 ARREIISAA  54 *CLP-2655IISAAEHFS  58 *CLP-2656 ISAAEHFSM  59  CLP-2657 SAAEHFSMI  60 *CLP-2659AEHFSMIRA  62 *CLP-2663 SMIRASRNK  66  CLP-2666 RASRNKSGA  69 *CLP-2670NKSGAAFGV  73 *CLP-2673 GAAFGVAPA  76 *CLP-2674 AAFGVAPAL  77 *CLP-2677GVAPALPGQ  80 *CLP-2678 VAPALPGQV  81 *CLP-2680 PALPGQVTI  83 *CLP-2681ALPGQVTIR  84 *CLP-2682 LPGQVTIRV  85  CLP-2684 GQVTIRVRV  87 *CLP-2689RVRVPYRVV  92 *CLP-2691 RVPYRVVGL  94 *CLP-2692 VPYRVVGLV  95 *CLP-2695RVVGLVVGP  98 *CLP-2698 GLVVGPKGA 101 *CLP-2699 LVVGPKGAT 102 *CLP-2700VVGPKGATI 103 *CLP-2710 RIQQQTNTY 113 *CLP-2711 IQQQTNTYI 114 *CLP-2712QQQTNTYII 115 *CLP-2713 QQTNTYIIT 116 *CLP-2718 YIITPSRDR 121  CLP-2721TPSRDRDPV 124  CLP-2724 RDRDPVFEI 127  CLP-2731 EITGAPGNV 134  CLP-2734GAPGNVERA 137  CLP-2738 NVERAREEI 141  CLP-2744 EEIETHIAV 147  CLP-2746IETHIAVRT 149  CLP-2749 HIAVRTGKI 152  CLP-2750 IAVRTGKIL 153  CLP-2756KILEYNNEN 159  CLP-2760 YNNENDFLA 163  CLP-2762 NENDFLAGS 165  CLP-2766FLAGSPDAA 169  CLP-2767 LAGSPDAAI 170  CLP-2774 AIDSRYSDA 177  CLP-2777SRYSDAWRV 180  CLP-2785 VHQPGCKPL 188  CLP-2793 LSTFRQNSL 196  CLP-2801LGCIGECGV 204  CLP-2807 CGVDSGFEA 210  CLP-2812 GFEAPRLDV 215  CLP-2817RLDVYYGVA 220  CLP-2819 DVYYGVAET 222  CLP-2823 GVAETSPPL 226  CLP-2825AETSPPLWA 228  CLP-2830 PLWAGQENA 233  CLP-2833 AGQENATPT 236  CLP-2835QENATPTSV 238  CLP-2843 VLFSSASSS 246  CLP-2857 KARAGPPGA 260  CLP-2869PATSAGPEL 272  CLP-2870 ATSAGPELA 273  CLP-2872 SAGPELAGL 275  CLP-2879GLPRRPPGE 282  CLP-2887 EPLQGFSKL 290  CLP-2892 FSKLGGGGL 295  CLP-2894KLGGGGLRS 297  CLP-2899 GLRSPGGGR 302  CLP-2909 CMVCFESEV 312  CLP-2910MVCFESEVT 313  CLP-2911 VCFESEVTA 314  CLP-2913 FESEVTAAL 316  CLP-2916EVTAALVPC 319  CLP-2917 VTAALVPCG 320  CLP-2920 ALVPCGHNL 323  CLP-2921LVPCGHNLF 324  CLP-2922 VPCGHNLFC 325  CLP-2927 NLFCMECAV 330  CLP-2929FCMECAVRI 332  CLP-2933 CAVRICERT 336  CLP-2936 RICERTDPE 339  CLP-2940RTDPECPVC 343  CLP-2945 CPVCHITAT 348  CLP-2947 VCHITATQA 350  CLP-2950ITATQAIRI 353

[0124] TABLE IX shows the groups of peptides used for immunologicaltesting: Peptide Peptide Peptide Group Number Sequence 1 CLP 2887EPLQGFSKL CLP 2916 EVTAALVPC CLP 2945 CPVCHITAT CLP 2673 KIKALRAKT CLP2699 IISAAEHFS CLP 2616 RASRNKSGA CLP 2655 GAAFGVAPA CLP 2731 LVVGPKGATCLP 2734 EITGAPGNV CLP 2666 GAPGNVERA 2 CLP 2724 ALRAKTNTY CLP 2689VATARREII CLP 2648 PALPGQVTI CLP 2680 ALPGQVTIR CLP 2619 RVRVPYRVV CLP2681 RDRDPVFEI CLP 2689 RVRVPYRVV CLP 2947 HIAVRTGKI CLP 2762 NENDFLAGSCLP 2933 CAVRICERT CLP 2749 VCHITATQA 3 CLP 2647 GRREDVATA CLP 2677DVATARREI CLP 2643 TARREIISA CLP 2785 GVAPALPGQ CLP 2917 RVVGLVVGP CLP2695 VHQPGCKPL CLP 2650 PATSAGPEL CLP 2869 VTAALVPCG 4 CLP 2812VPVPTSEHV CLP 2892 ARREIISAA CLP 2738 RIQQQTNTY CLP 2651 NVERAREEI CLP2870 GFEAPRLDV CLP 2899 ATSAGPELA CLP 2710 FSKLGGGGL CLP 2599 GLRSPGGGR5 CLP 2609 PTSEHVAEI CLP 2602 EIVGRQCKI CLP 2641 LRAKTNTYI CLP 2620VTGRREDVA CLP 2940 SMIRASRNK CLP 2921 CMVCFESEV CLP 2936 LVPCGHNLF CLP2663 NLFCMECAV CLP 2927 RICERTDPE CLP 2909 RTDPECPVC 6 CLP 2766MVTGRREDV CLP 2711 GLVVGPKGA CLP 2913 IQQQTNTYI CLP 2823 FLAGSPDAA CLP2640 GVAETSPPL CLP 2698 FESEVTAAL CLP 2929 FCMECAVRI 7 CLP 2760KALRAKTNT CLP 2633 RGEEPVFMV CLP 2700 SAAEHFSMI CLP 2835 AAFGVAPAL CLP2618 VVGPKGATI CLP 2657 YNNENDFLA CLP 2674 LGCIGECGV CLP 2911 QENATPTSVCLP 2801 VCFESEVTA 8 CLP 2807 TNTYIKTPV CLP 2872 NKSGAAFGV CLP 2670QQTNTYIIT CLP 2756 KILEYNNEN CLP 2825 CGVDSGFEA CLP 2843 AETSPPLWA CLP2713 PLWAGQENA CLP 2624 VLFSSASSS CLP 2830 SAGPELAGL 9 CLP 2712ISAAEHFSM CLP 2744 QQQTNTYII CLP 2774 EEIETHIAV CLP 2819 IETHIAVRT CLP2656 LAGSPDAAI CLP 2922 AIDSRYSDA CLP 2746 DVYYGVAET CLP 2767 VPCGHNLFCCLP 2950 ITATQAIRI 10 CLP 2793 TPVRGEEPV CLP 2777 AEHFSMIRA CLP 2910VAPALPGQV CLP 2721 TPSRDRDPV CLP 2630 IAVRTGKIL CLP 2659 SRYSDAWRV CLP2678 LSTFRQNSL CLP 2750 RLDVYYGVA CLP 2833 AGQENATPT CLP 2817 MVCFESEVT

[0125] C. Immune Reactivity of BCY1 Peptides and Generation of HumanEffector T Cells:

[0126] The library of 100 peptides from BCY1 was separated into 10groups of 7-10 peptides for immunological testing. Dissolved peptidepools were pulsed onto autologous HLA-A*0201 dendritic cells and used toactivate autologous T-cell-enriched PBMC preparations. Activated T cellsfrom each peptide-pool-stimulated culture were re-stimulated another 3to 5 times using CD40L-activated autologous B-cells. IFN-γ ELISPOTanalysis and assays for CTL killing of peptide-pulsed target cells wasperformed to demonstrate the immunogenicity of these epitopes from BCY1.

[0127] Human T cells demonstrated effector cell activity against anumber of pools of peptides from the BCY1 protein, as shown by theirability to secrete IFN-γ in ELISPOT assays. These experiments wererepeated after different rounds of APC stimulation resulting in the samereactive peptide groups. Peptide groups 1, 2, 3, 4, 5, 6, and 7 werefound to be immunoreactive in these assays. Subsequently, these reactivepeptide groups were de-convoluted in additional IFN-γ ELISPOT assays inwhich single peptides from each group were tested separately. Thisanalysis revealed a number of individual strongly reactive peptides fromthe BCY1 protein recognized by human T cells. Many of these singlepeptides also induced CTL activity killing peptide-loaded human T2lymphoma cell targets. Table IX lists these peptides.

[0128] While the present invention has been described in terms of thepreferred embodiments, it is understood that variations andmodifications will occur to those skilled in the art. Therefore, it isintended that the appended claims cover all such equivalent variationsthat come within the scope of the invention as claimed.

1 218 1 3846 DNA Homo sapiens 1 atggtccgga aaaagaaccc ccctctgagaaacgttgcaa gtgaaggcga gggccagatc 60 ctggagccta taggtacaga aagcaaggtatctggaaaga acaaagaatt ctctgcagat 120 cagatgtcag aaaatacgga tcagagtgatgctgcagaac taaatcataa ggaggaacat 180 agcttgcatg ttcaagatcc atcttctagcagtaagaagg acttgaaaag cgcagttctg 240 agtgagaagg ctggcttcaa ttatgaaagccccagtaagg gaggaaactt tccctccttt 300 ccgcatgatg aggtgacaga cagaaatatgttggctttct catttccagc tgctggggga 360 gtctgtgagc ccttgaagtc tccgcaaagagcagaggcag atgaccctca agatatggcc 420 tgcaccccct caggggactc actggagacaaaggaagatc agaagatgtc accaaaggct 480 acagaggaaa cagggcaagc acagagtggtcaagccaatt gtcaaggttt gagcccagtt 540 tcagtggcct caaaaaaccc acaagtgccttcagatgggg gtgtaagact gaataaatcc 600 aaaactgact tactggtgaa tgacaacccagacccggcac ctctgtctcc agagcttcag 660 gactttaaat gcaatatctg tggatatggttactacggca acgaccccac agatctgatt 720 aagcacttcc gaaagtatca cttaggactgcataaccgca ccaggcaaga tgctgagctg 780 gacagcaaaa tcttggccct tcataacatggtgcagttca gccattccaa agacttccag 840 aaggtcaacc gttctgtgtt ttctggtgtgctgcaggaca tcaattcttc aaggcctgtt 900 ttactaaatg ggacctatga tgtgcaggtgacttcaggtg gaacattcat tggcattgga 960 cggaaaacac cagattgcca agggaacaccaagtatttcc gctgtaaatt ctgcaatttc 1020 acttatatgg gcaactcatc caccgaattagaacaacatt ttcttcagac tcacccaaac 1080 aaaataaaag cttctctccc ctcctctgaggttgcaaaac cttcagagaa aaactctaac 1140 aagtccatcc ctgcacttca atccagtgattctggagact tgggaaaatg gcaggacaag 1200 ataacagtca aagcaggaga tgacactcctgttgggtact cagtgcccat aaagcccctc 1260 gattcctcta gacaaaatgg tacagaggccaccagttact actggtgtaa attttgtagt 1320 ttcagctgtg agtcatctag ctcacttaaactgctagaac attatggcaa gcagcacgga 1380 gcagtgcagt caggcggcct taatccagagttaaatgata agctttccag gggctctgtc 1440 attaatcaga atgatctagc caaaagttcagaaggagaga caatgaccaa gacagacaag 1500 agctcgagtg gggctaaaaa gaaggacttctccagcaagg gagccgagga taatatggta 1560 acgagctata attgtcagtt ctgtgacttccgatattcca aaagccatgg ccctgatgta 1620 attgtagtgg ggccacttct ccgtcattatcaacagctcc ataacattca caagtgtacc 1680 attaaacact gtccattctg tcccagaggactttgcagcc cagaaaagca ccttggagaa 1740 attacttatc cgtttgcttg tagaaaaagtaattgttccc actgtgcact cttgcttctg 1800 cacttgtctc ctggggcggc tggaagctcgcgagtcaaac atcagtgcca tcagtgttca 1860 ttcaccaccc ctgacgtaga tgtactcctctttcactatg aaagtgtgca tgagtcccaa 1920 gcatcggatg tcaaacaaga agcaaatcacctgcaaggat cggatgggca gcagtctgtc 1980 aaggaaagca aagaacactc atgtaccaaatgtgatttta ttacccaagt ggaagaagag 2040 atttcccgac actacaggag agcacacagctgctacaaat gccgtcagtg cagttttaca 2100 gctgccgata ctcagtcact actggagcacttcaacactg ttcactgcca ggaacaggac 2160 atcactacag ccaacggcga agaggacggtcatgccatat ccaccatcaa agaggagccc 2220 aaaattgact tcagggtcta caatctgctaactccagact ctaaaatggg agagccagtt 2280 tctgagagtg tggtgaagag agagaagctggaagagaagg acgggctcaa agagaaagtt 2340 tggaccgaga gttccagtga tgaccttcgcaatgtgactt ggagaggggc agacatcctg 2400 cgggggagtc cgtcatacac ccaagcaagcctggggctgc tgacgcctgt gtctggcacc 2460 caagagcaga caaagactct aagggatagtcccaatgtgg aggccgccca tctggcgcga 2520 cctatttatg gcttggctgt ggaaaccaagggattcctgc agggggcgcc agctggcgga 2580 gagaagtctg gggccctccc ccagcagtatcctgcatcgg gagaaaacaa gtccaaggat 2640 gaatcccagt ccctgttacg gaggcgtagaggctccggtg ttttttgtgc caattgcctg 2700 accacaaaga cctctctctg gcgaaagaatgcaaatggcg gatatgtatg caacgcgtgt 2760 ggcctctacc agaagcttca ctcgactcccaggcctttaa acatcattaa acaaaacaac 2820 ggtgagcaga ttattaggag gagaacaagaaagcgcctta acccagaggc acttcaggct 2880 gagcagctca acaaacagca gaggggcagcaatgaggagc aagtcaatgg aagcccgtta 2940 gagaggaggt cagaagatca tctaactgaaagtcaccaga gagaaattcc actccccagc 3000 ctaagtaaat acgaagccca gggttcattgactaaaagcc attctgctca gcagccagtc 3060 ctggtcagcc aaactctgga tattcacaaaaggatgcaac ctttgcacat tcagataaaa 3120 agtcctcagg aaagtactgg agatccaggaaatagttcat ccgtatctga agggaaagga 3180 agttctgaga gaggcagtcc tatagaaaagtacatgagac ctgcgaaaca cccaaattat 3240 tcaccaccag gcagccctat tgaaaagtaccagtacccac tttttggact tccctttgta 3300 cataatgact tccagagtga agctgattggctgcggttct ggagtaaata taagctctcc 3360 gttcctggga atccgcacta cttgagtcacgtgcctggcc taccaaatcc ttgccaaaac 3420 tatgtgcctt atcccacctt caatctgcctcctcattttt cagctgttgg atcagacaat 3480 gacattcctc tagatttggc gatcaagcattccagacctg ggccaactgc aaacggtgcc 3540 tccaaggaga aaacgaaggc accaccaaatgtaaaaaatg aaggtccctt gaatgtagta 3600 aaaacagaga aagttgatag aagtactcaagatgaacttt caacaaaatg tgtgcactgt 3660 ggcattgtct ttctggatga agtgatgtatgctttgcata tgagttgcca tggtgacagt 3720 ggacctttcc agtgcagcat atgccagcatctttgcacgg acaaatatga cttcacaaca 3780 catatccaga ggggcctgca taggaacaatgcacaagtgg aaaaaaatgg aaaacctaaa 3840 gagtaa 3846 2 1281 PRT Homosapiens 2 Met Val Arg Lys Lys Asn Pro Pro Leu Arg Asn Val Ala Ser GluGly 1 5 10 15 Glu Gly Gln Ile Leu Glu Pro Ile Gly Thr Glu Ser Lys ValSer Gly 20 25 30 Lys Asn Lys Glu Phe Ser Ala Asp Gln Met Ser Glu Asn ThrAsp Gln 35 40 45 Ser Asp Ala Ala Glu Leu Asn His Lys Glu Glu His Ser LeuHis Val 50 55 60 Gln Asp Pro Ser Ser Ser Ser Lys Lys Asp Leu Lys Ser AlaVal Leu 65 70 75 80 Ser Glu Lys Ala Gly Phe Asn Tyr Glu Ser Pro Ser LysGly Gly Asn 85 90 95 Phe Pro Ser Phe Pro His Asp Glu Val Thr Asp Arg AsnMet Leu Ala 100 105 110 Phe Ser Phe Pro Ala Ala Gly Gly Val Cys Glu ProLeu Lys Ser Pro 115 120 125 Gln Arg Ala Glu Ala Asp Asp Pro Gln Asp MetAla Cys Thr Pro Ser 130 135 140 Gly Asp Ser Leu Glu Thr Lys Glu Asp GlnLys Met Ser Pro Lys Ala 145 150 155 160 Thr Glu Glu Thr Gly Gln Ala GlnSer Gly Gln Ala Asn Cys Gln Gly 165 170 175 Leu Ser Pro Val Ser Val AlaSer Lys Asn Pro Gln Val Pro Ser Asp 180 185 190 Gly Gly Val Arg Leu AsnLys Ser Lys Thr Asp Leu Leu Val Asn Asp 195 200 205 Asn Pro Asp Pro AlaPro Leu Ser Pro Glu Leu Gln Asp Phe Lys Cys 210 215 220 Asn Ile Cys GlyTyr Gly Tyr Tyr Gly Asn Asp Pro Thr Asp Leu Ile 225 230 235 240 Lys HisPhe Arg Lys Tyr His Leu Gly Leu His Asn Arg Thr Arg Gln 245 250 255 AspAla Glu Leu Asp Ser Lys Ile Leu Ala Leu His Asn Met Val Gln 260 265 270Phe Ser His Ser Lys Asp Phe Gln Lys Val Asn Arg Ser Val Phe Ser 275 280285 Gly Val Leu Gln Asp Ile Asn Ser Ser Arg Pro Val Leu Leu Asn Gly 290295 300 Thr Tyr Asp Val Gln Val Thr Ser Gly Gly Thr Phe Ile Gly Ile Gly305 310 315 320 Arg Lys Thr Pro Asp Cys Gln Gly Asn Thr Lys Tyr Phe ArgCys Lys 325 330 335 Phe Cys Asn Phe Thr Tyr Met Gly Asn Ser Ser Thr GluLeu Glu Gln 340 345 350 His Phe Leu Gln Thr His Pro Asn Lys Ile Lys AlaSer Leu Pro Ser 355 360 365 Ser Glu Val Ala Lys Pro Ser Glu Lys Asn SerAsn Lys Ser Ile Pro 370 375 380 Ala Leu Gln Ser Ser Asp Ser Gly Asp LeuGly Lys Trp Gln Asp Lys 385 390 395 400 Ile Thr Val Lys Ala Gly Asp AspThr Pro Val Gly Tyr Ser Val Pro 405 410 415 Ile Lys Pro Leu Asp Ser SerArg Gln Asn Gly Thr Glu Ala Thr Ser 420 425 430 Tyr Tyr Trp Cys Lys PheCys Ser Phe Ser Cys Glu Ser Ser Ser Ser 435 440 445 Leu Lys Leu Leu GluHis Tyr Gly Lys Gln His Gly Ala Val Gln Ser 450 455 460 Gly Gly Leu AsnPro Glu Leu Asn Asp Lys Leu Ser Arg Gly Ser Val 465 470 475 480 Ile AsnGln Asn Asp Leu Ala Lys Ser Ser Glu Gly Glu Thr Met Thr 485 490 495 LysThr Asp Lys Ser Ser Ser Gly Ala Lys Lys Lys Asp Phe Ser Ser 500 505 510Lys Gly Ala Glu Asp Asn Met Val Thr Ser Tyr Asn Cys Gln Phe Cys 515 520525 Asp Phe Arg Tyr Ser Lys Ser His Gly Pro Asp Val Ile Val Val Gly 530535 540 Pro Leu Leu Arg His Tyr Gln Gln Leu His Asn Ile His Lys Cys Thr545 550 555 560 Ile Lys His Cys Pro Phe Cys Pro Arg Gly Leu Cys Ser ProGlu Lys 565 570 575 His Leu Gly Glu Ile Thr Tyr Pro Phe Ala Cys Arg LysSer Asn Cys 580 585 590 Ser His Cys Ala Leu Leu Leu Leu His Leu Ser ProGly Ala Ala Gly 595 600 605 Ser Ser Arg Val Lys His Gln Cys His Gln CysSer Phe Thr Thr Pro 610 615 620 Asp Val Asp Val Leu Leu Phe His Tyr GluSer Val His Glu Ser Gln 625 630 635 640 Ala Ser Asp Val Lys Gln Glu AlaAsn His Leu Gln Gly Ser Asp Gly 645 650 655 Gln Gln Ser Val Lys Glu SerLys Glu His Ser Cys Thr Lys Cys Asp 660 665 670 Phe Ile Thr Gln Val GluGlu Glu Ile Ser Arg His Tyr Arg Arg Ala 675 680 685 His Ser Cys Tyr LysCys Arg Gln Cys Ser Phe Thr Ala Ala Asp Thr 690 695 700 Gln Ser Leu LeuGlu His Phe Asn Thr Val His Cys Gln Glu Gln Asp 705 710 715 720 Ile ThrThr Ala Asn Gly Glu Glu Asp Gly His Ala Ile Ser Thr Ile 725 730 735 LysGlu Glu Pro Lys Ile Asp Phe Arg Val Tyr Asn Leu Leu Thr Pro 740 745 750Asp Ser Lys Met Gly Glu Pro Val Ser Glu Ser Val Val Lys Arg Glu 755 760765 Lys Leu Glu Glu Lys Asp Gly Leu Lys Glu Lys Val Trp Thr Glu Ser 770775 780 Ser Ser Asp Asp Leu Arg Asn Val Thr Trp Arg Gly Ala Asp Ile Leu785 790 795 800 Arg Gly Ser Pro Ser Tyr Thr Gln Ala Ser Leu Gly Leu LeuThr Pro 805 810 815 Val Ser Gly Thr Gln Glu Gln Thr Lys Thr Leu Arg AspSer Pro Asn 820 825 830 Val Glu Ala Ala His Leu Ala Arg Pro Ile Tyr GlyLeu Ala Val Glu 835 840 845 Thr Lys Gly Phe Leu Gln Gly Ala Pro Ala GlyGly Glu Lys Ser Gly 850 855 860 Ala Leu Pro Gln Gln Tyr Pro Ala Ser GlyGlu Asn Lys Ser Lys Asp 865 870 875 880 Glu Ser Gln Ser Leu Leu Arg ArgArg Arg Gly Ser Gly Val Phe Cys 885 890 895 Ala Asn Cys Leu Thr Thr LysThr Ser Leu Trp Arg Lys Asn Ala Asn 900 905 910 Gly Gly Tyr Val Cys AsnAla Cys Gly Leu Tyr Gln Lys Leu His Ser 915 920 925 Thr Pro Arg Pro LeuAsn Ile Ile Lys Gln Asn Asn Gly Glu Gln Ile 930 935 940 Ile Arg Arg ArgThr Arg Lys Arg Leu Asn Pro Glu Ala Leu Gln Ala 945 950 955 960 Glu GlnLeu Asn Lys Gln Gln Arg Gly Ser Asn Glu Glu Gln Val Asn 965 970 975 GlySer Pro Leu Glu Arg Arg Ser Glu Asp His Leu Thr Glu Ser His 980 985 990Gln Arg Glu Ile Pro Leu Pro Ser Leu Ser Lys Tyr Glu Ala Gln Gly 995 10001005 Ser Leu Thr Lys Ser His Ser Ala Gln Gln Pro Val Leu Val Ser 10101015 1020 Gln Thr Leu Asp Ile His Lys Arg Met Gln Pro Leu His Ile Gln1025 1030 1035 Ile Lys Ser Pro Gln Glu Ser Thr Gly Asp Pro Gly Asn SerSer 1040 1045 1050 Ser Val Ser Glu Gly Lys Gly Ser Ser Glu Arg Gly SerPro Ile 1055 1060 1065 Glu Lys Tyr Met Arg Pro Ala Lys His Pro Asn TyrSer Pro Pro 1070 1075 1080 Gly Ser Pro Ile Glu Lys Tyr Gln Tyr Pro LeuPhe Gly Leu Pro 1085 1090 1095 Phe Val His Asn Asp Phe Gln Ser Glu AlaAsp Trp Leu Arg Phe 1100 1105 1110 Trp Ser Lys Tyr Lys Leu Ser Val ProGly Asn Pro His Tyr Leu 1115 1120 1125 Ser His Val Pro Gly Leu Pro AsnPro Cys Gln Asn Tyr Val Pro 1130 1135 1140 Tyr Pro Thr Phe Asn Leu ProPro His Phe Ser Ala Val Gly Ser 1145 1150 1155 Asp Asn Asp Ile Pro LeuAsp Leu Ala Ile Lys His Ser Arg Pro 1160 1165 1170 Gly Pro Thr Ala AsnGly Ala Ser Lys Glu Lys Thr Lys Ala Pro 1175 1180 1185 Pro Asn Val LysAsn Glu Gly Pro Leu Asn Val Val Lys Thr Glu 1190 1195 1200 Lys Val AspArg Ser Thr Gln Asp Glu Leu Ser Thr Lys Cys Val 1205 1210 1215 His CysGly Ile Val Phe Leu Asp Glu Val Met Tyr Ala Leu His 1220 1225 1230 MetSer Cys His Gly Asp Ser Gly Pro Phe Gln Cys Ser Ile Cys 1235 1240 1245Gln His Leu Cys Thr Asp Lys Tyr Asp Phe Thr Thr His Ile Gln 1250 12551260 Arg Gly Leu His Arg Asn Asn Ala Gln Val Glu Lys Asn Gly Lys 12651270 1275 Pro Lys Glu 1280 3 1203 DNA Homo sapiens 3 atggccgagctgcgcctgaa gggcagcagc aacaccacgg agtgtgttcc cgtgcccacc 60 tccgagcacgtggccgagat cgtgggcagg caaggctgca agattaaggc cttgagggcc 120 aagaccaacacctacatcaa gacaccggtg aggggcgagg aaccagtgtt catggtgaca 180 gggcgacgggaggacgtggc cacagcccgg cgggaaatca tctcagcagc ggagcacttc 240 tccatgatccgtgcctcccg caacaagtca ggcgccgcct ttggtgtggc tcctgctctg 300 cccggccaggtgaccatccg tgtgcgggtg ccctaccgcg tggtggggct ggtggtgggc 360 cccaaaggggcaaccatcaa gcgcatccag cagcaaacca acacatacat tatcacacca 420 agccgtgaccgcgaccccgt gttcgagatc acgggtgccc caggcaacgt ggagcgtgcg 480 cgcgaggagatcgagacgca catcgcggtg cgcactggca agatcctcga gtacaacaat 540 gaaaacgacttcctggcggg gagccccgac gcagcaatcg atagccgcta ctccgacgcc 600 tggcgggtgcaccagcccgg ctgcaagccc ctctccacct tccggcagaa cagcctgggc 660 tgcatcggcgagtgcggagt ggactctggc tttgaggccc cacgcctggg tgagcagggc 720 ggggactttggctacggcgg gtacctcttt ccgggctatg gcgtgggcaa gcaggatgtg 780 tactacggcgtggccgagac tagccccccg ctgtgggcgg gccaggagaa cgccacgccc 840 acctccgtgctcttctcctc tgcctcctcc tcctcctcct cttccgccaa ggcccgcgct 900 gggcccccgggcgcacaccg ctcccctgcc acttccgcgg gacccgagct ggccggactc 960 ccgaggcgccccccgggaga gccgctccag ggcttctcta aacttggtgg gggcggcctg 1020 cggagccccggcggcgggcg ggattgcatg gtctgctttg agagcgaagt gactgccgcc 1080 cttgtgccctgcggacacaa cctgttctgc atggagtgtg cagtacgcat ctgcgagagg 1140 acggacccagagtgtcccgt ctgccacatc acagccgcgc aagccatccg aatattctcc 1200 taa 1203 4400 PRT Homo sapiens 4 Met Ala Glu Leu Arg Leu Lys Gly Ser Ser Asn ThrThr Glu Cys Val 1 5 10 15 Pro Val Pro Thr Ser Glu His Val Ala Glu IleVal Gly Arg Gln Gly 20 25 30 Cys Lys Ile Lys Ala Leu Arg Ala Lys Thr AsnThr Tyr Ile Lys Thr 35 40 45 Pro Val Arg Gly Glu Glu Pro Val Phe Met ValThr Gly Arg Arg Glu 50 55 60 Asp Val Ala Thr Ala Arg Arg Glu Ile Ile SerAla Ala Glu His Phe 65 70 75 80 Ser Met Ile Arg Ala Ser Arg Asn Lys SerGly Ala Ala Phe Gly Val 85 90 95 Ala Pro Ala Leu Pro Gly Gln Val Thr IleArg Val Arg Val Pro Tyr 100 105 110 Arg Val Val Gly Leu Val Val Gly ProLys Gly Ala Thr Ile Lys Arg 115 120 125 Ile Gln Gln Gln Thr Asn Thr TyrIle Ile Thr Pro Ser Arg Asp Arg 130 135 140 Asp Pro Val Phe Glu Ile ThrGly Ala Pro Gly Asn Val Glu Arg Ala 145 150 155 160 Arg Glu Glu Ile GluThr His Ile Ala Val Arg Thr Gly Lys Ile Leu 165 170 175 Glu Tyr Asn AsnGlu Asn Asp Phe Leu Ala Gly Ser Pro Asp Ala Ala 180 185 190 Ile Asp SerArg Tyr Ser Asp Ala Trp Arg Val His Gln Pro Gly Cys 195 200 205 Lys ProLeu Ser Thr Phe Arg Gln Asn Ser Leu Gly Cys Ile Gly Glu 210 215 220 CysGly Val Asp Ser Gly Phe Glu Ala Pro Arg Leu Gly Glu Gln Gly 225 230 235240 Gly Asp Phe Gly Tyr Gly Gly Tyr Leu Phe Pro Gly Tyr Gly Val Gly 245250 255 Lys Gln Asp Val Tyr Tyr Gly Val Ala Glu Thr Ser Pro Pro Leu Trp260 265 270 Ala Gly Gln Glu Asn Ala Thr Pro Thr Ser Val Leu Phe Ser SerAla 275 280 285 Ser Ser Ser Ser Ser Ser Ser Ala Lys Ala Arg Ala Gly ProPro Gly 290 295 300 Ala His Arg Ser Pro Ala Thr Ser Ala Gly Pro Glu LeuAla Gly Leu 305 310 315 320 Pro Arg Arg Pro Pro Gly Glu Pro Leu Gln GlyPhe Ser Lys Leu Gly 325 330 335 Gly Gly Gly Leu Arg Ser Pro Gly Gly GlyArg Asp Cys Met Val Cys 340 345 350 Phe Glu Ser Glu Val Thr Ala Ala LeuVal Pro Cys Gly His Asn Leu 355 360 365 Phe Cys Met Glu Cys Ala Val ArgIle Cys Glu Arg Thr Asp Pro Glu 370 375 380 Cys Pro Val Cys His Ile ThrAla Ala Gln Ala Ile Arg Ile Phe Ser 385 390 395 400 5 22 PRT ArtificialCLP 2589 5 Met Val Arg Lys Lys Asn Pro Pro Leu Arg Asn Val Ala Ser GluGly 1 5 10 15 Glu Gly Gln Ile Leu Glu 20 6 22 PRT Artificial CLP 2590 6Ser Pro Lys Ala Thr Glu Glu Thr Gly Gln Ala Gln Ser Gly Gln Ala 1 5 1015 Asn Cys Gln Gly Leu Ser 20 7 22 PRT Artificial CLP 2591 7 Val Ala LysPro Ser Glu Lys Asn Ser Asn Lys Ser Ile Pro Ala Leu 1 5 10 15 Gln SerSer Asp Ser Gly 20 8 22 PRT Artificial CLP 2592 8 Asn His Leu Gln GlySer Asp Gly Gln Gln Ser Val Lys Glu Ser Lys 1 5 10 15 Glu His Ser CysThr Lys 20 9 22 PRT Artificial CLP 2593 9 Asn Gly Glu Gln Ile Ile ArgArg Arg Thr Arg Lys Arg Leu Asn Pro 1 5 10 15 Glu Ala Leu Gln Ala Glu 2010 23 PRT Artificial CLP 2594 10 Ala Asn Gly Ala Ser Lys Glu Lys Thr LysAla Pro Pro Asn Val Lys 1 5 10 15 Asn Glu Gly Pro Leu Asn Val 20 11 32DNA Artificial Primer 7717 11 cgggatccac catggtccgg aaaaagaacc cc 32 1235 DNA Artificial Primer 7723 12 cgggatccct ctttaggttt tccatttttt tccac35 13 9 PRT Artificial CLP-2421 13 Met Val Arg Lys Lys Asn Pro Pro Leu 15 14 9 PRT Artificial CLP-2422 14 Lys Lys Asn Pro Pro Leu Arg Asn Val 15 15 9 PRT Artificial CLP-2423 15 Val Ala Ser Glu Gly Glu Gly Gln Ile 15 16 9 PRT Artificial CLP-2424 16 Gln Ile Leu Glu Pro Ile Gly Thr Glu 15 17 9 PRT Artificial CLP-2425 17 Arg Asn Met Leu Ala Phe Ser Phe Pro 15 18 9 PRT Artificial CLP-2426 18 Asn Met Leu Ala Phe Ser Phe Pro Ala 15 19 9 PRT Artificial CLP-2427 19 Met Leu Ala Phe Ser Phe Pro Ala Ala 15 20 9 PRT Artificial CLP-2428 20 Phe Ser Phe Pro Ala Ala Gly Gly Val 15 21 9 PRT Artificial CLP-2429 21 Ala Ala Gly Gly Val Cys Glu Pro Leu 15 22 9 PRT Artificial CLP-2430 22 Ser Gly Gln Ala Asn Cys Gln Gly Leu 15 23 9 PRT Artificial CLP-2431 23 Ala Asn Cys Gln Gly Leu Ser Pro Val 15 24 9 PRT Artificial CLP-2432 24 Gly Leu Ser Pro Val Ser Val Ala Ser 15 25 9 PRT Artificial CLP-2433 25 Ser Val Ala Ser Lys Asn Pro Gln Val 15 26 9 PRT Artificial CLP-2434 26 Arg Leu Asn Lys Ser Lys Thr Asp Leu 15 27 9 PRT Artificial CLP-2435 27 Asn Asp Asn Pro Asp Pro Ala Pro Leu 15 28 9 PRT Artificial CLP-2436 28 Asp Pro Ala Pro Leu Ser Pro Glu Leu 15 29 9 PRT Artificial CLP-2437 29 Glu Leu Gln Asp Phe Lys Cys Asn Ile 15 30 9 PRT Artificial CLP-2438 30 Gly Leu His Asn Arg Thr Arg Gln Asp 15 31 9 PRT Artificial CLP-2439 31 Glu Leu Asp Ser Lys Ile Leu Ala Leu 15 32 9 PRT Artificial CLP-2440 32 Lys Ile Leu Ala Leu His Asn Met Val 15 33 9 PRT Artificial CLP-2441 33 Ala Leu His Asn Met Val Gln Phe Ser 15 34 9 PRT Artificial CLP-2442 34 Val Asn Arg Ser Val Phe Ser Gly Val 15 35 9 PRT Artificial CLP-2443 35 Phe Ser Gly Val Leu Gln Asp Ile Asn 15 36 9 PRT Artificial CLP-2444 36 Asp Ile Asn Ser Ser Arg Pro Val Leu 15 37 9 PRT Artificial CLP-2445 37 Val Leu Leu Asn Gly Thr Tyr Asp Val 15 38 9 PRT Artificial CLP-2446 38 Phe Cys Asn Phe Thr Tyr Met Gly Asn 15 39 9 PRT Artificial CLP-2447 39 Tyr Met Gly Asn Ser Ser Thr Glu Leu 15 40 9 PRT Artificial CLP-2448 40 Phe Leu Gln Thr His Pro Asn Lys Ile 15 41 9 PRT Artificial CLP-2449 41 Lys Ala Ser Leu Pro Ser Ser Glu Val 15 42 9 PRT Artificial CLP-2450 42 Asp Leu Gly Lys Trp Gln Asp Lys Ile 15 43 9 PRT Artificial CLP-2451 43 Val Lys Ala Gly Asp Asp Thr Pro Val 15 44 9 PRT Artificial CLP-2452 44 Phe Ser Cys Glu Ser Ser Ser Ser Leu 15 45 9 PRT Artificial CLP-2453 45 Lys Leu Leu Glu His Tyr Gly Lys Gln 15 46 9 PRT Artificial CLP-2454 46 Gly Leu Asn Pro Glu Leu Asn Asp Lys 15 47 9 PRT Artificial CLP-2455 47 Gly Ser Val Ile Asn Gln Asn Asp Leu 15 48 9 PRT Artificial CLP-2456 48 Ser Val Ile Asn Gln Asn Asp Leu Ala 15 49 9 PRT Artificial CLP-2457 49 Phe Cys Asp Phe Arg Tyr Ser Lys Ser 15 50 9 PRT Artificial CLP-2458 50 Ser His Gly Pro Asp Val Ile Val Val 15 51 9 PRT Artificial CLP-2459 51 Pro Leu Leu Arg His Tyr Gln Gln Leu 15 52 9 PRT Artificial CLP-2460 52 Gly Leu Cys Ser Pro Glu Lys His Leu 15 53 9 PRT Artificial CLP-2461 53 His Leu Gly Glu Ile Thr Tyr Pro Phe 15 54 9 PRT Artificial CLP-2462 54 Leu Gly Glu Ile Thr Tyr Pro Phe Ala 15 55 9 PRT Artificial CLP-2463 55 His Cys Ala Leu Leu Leu Leu His Leu 15 56 9 PRT Artificial CLP-2464 56 Ala Leu Leu Leu Leu His Leu Ser Pro 15 57 9 PRT Artificial CLP-2465 57 Leu Leu Leu Leu His Leu Ser Pro Gly 15 58 9 PRT Artificial CLP-2466 58 Leu Leu Leu His Leu Ser Pro Gly Ala 15 59 9 PRT Artificial CLP-2467 59 Leu Leu His Leu Ser Pro Gly Ala Ala 15 60 9 PRT Artificial CLP-2468 60 Phe Thr Thr Pro Asp Val Asp Val Leu 15 61 9 PRT Artificial CLP-2469 61 Thr Thr Pro Asp Val Asp Val Leu Leu 15 62 9 PRT Artificial CLP-2470 62 Val Leu Leu Phe His Tyr Glu Ser Val 15 63 9 PRT Artificial CLP-2471 63 Phe Ile Thr Gln Val Glu Glu Glu Ile 15 64 9 PRT Artificial CLP-2472 64 Phe Thr Ala Ala Asp Thr Gln Ser Leu 15 65 9 PRT Artificial CLP-2473 65 Ser Leu Leu Glu His Phe Asn Thr Val 15 66 9 PRT Artificial CLP-2474 66 Ser Thr Ile Lys Glu Glu Pro Lys Ile 15 67 9 PRT Artificial CLP-2475 67 Lys Ile Asp Phe Arg Val Tyr Asn Leu 15 68 9 PRT Artificial CLP-2476 68 Asn Leu Leu Thr Pro Asp Ser Lys Met 15 69 9 PRT Artificial CLP-2479 69 Val Thr Trp Arg Gly Ala Asp Ile Leu 15 70 9 PRT Artificial CLP-2480 70 Ile Leu Arg Gly Ser Pro Ser Tyr Thr 15 71 9 PRT Artificial CLP-2481 71 Tyr Thr Gln Ala Ser Leu Gly Leu Leu 15 72 9 PRT Artificial CLP-2482 72 Ala Ser Leu Gly Leu Leu Thr Pro Val 15 73 9 PRT Artificial CLP-2483 73 Gly Leu Leu Thr Pro Val Ser Gly Thr 15 74 9 PRT Artificial CLP-2484 74 Gly Thr Gln Glu Gln Thr Lys Thr Leu 15 75 9 PRT Artificial CLP-2485 75 Lys Thr Leu Arg Asp Ser Pro Asn Val 15 76 9 PRT Artificial CLP-2486 76 His Leu Ala Arg Pro Ile Tyr Gly Leu 15 77 9 PRT Artificial CLP-2487 77 Pro Ile Tyr Gly Leu Ala Val Glu Thr 15 78 9 PRT Artificial CLP-2488 78 Leu Ala Val Glu Thr Lys Gly Phe Leu 15 79 9 PRT Artificial CLP-2489 79 Phe Leu Gln Gly Ala Pro Ala Gly Gly 15 80 9 PRT Artificial CLP-2490 80 Ala Gly Gly Glu Lys Ser Gly Ala Leu 15 81 9 PRT Artificial CLP-2491 81 Gly Ala Leu Pro Gln Gln Tyr Pro Ala 15 82 9 PRT Artificial CLP-2492 82 Ala Leu Pro Gln Gln Tyr Pro Ala Ser 15 83 9 PRT Artificial CLP-2493 83 Phe Cys Ala Asn Cys Leu Thr Thr Lys 15 84 9 PRT Artificial CLP-2494 84 Ala Asn Gly Gly Tyr Val Cys Asn Ala 15 85 9 PRT Artificial CLP-2495 85 Asn Ala Cys Gly Leu Tyr Gln Lys Leu 15 86 9 PRT Artificial CLP-2496 86 Gly Leu Tyr Gln Lys Leu His Ser Thr 15 87 9 PRT Artificial CLP-2497 87 Lys Leu His Ser Thr Pro Arg Pro Leu 15 88 9 PRT Artificial CLP-2498 88 Ser Thr Pro Arg Pro Leu Asn Ile Ile 15 89 9 PRT Artificial CLP-2499 89 Arg Leu Asn Pro Glu Ala Leu Gln Ala 15 90 9 PRT Artificial CLP-2500 90 Val Leu Val Ser Gln Thr Leu Asp Ile 15 91 9 PRT Artificial CLP-2501 91 Asp Ile His Lys Arg Met Gln Pro Leu 15 92 9 PRT Artificial CLP-2502 92 Arg Met Gln Pro Leu His Ile Gln Ile 15 93 9 PRT Artificial CLP-2503 93 Tyr Pro Leu Phe Gly Leu Pro Phe Val 15 94 9 PRT Artificial CLP-2504 94 Gly Leu Pro Phe Val His Asn Asp Phe 15 95 9 PRT Artificial CLP-2505 95 Phe Val His Asn Asp Phe Gln Ser Glu 15 96 9 PRT Artificial CLP-2506 96 Ser Val Pro Gly Asn Pro His Tyr Leu 15 97 9 PRT Artificial CLP-2507 97 Gly Asn Pro His Tyr Leu Ser His Val 15 98 9 PRT Artificial CLP-2508 98 His Tyr Leu Ser His Val Pro Gly Leu 15 99 9 PRT Artificial CLP-2509 99 Tyr Val Pro Tyr Pro Thr Phe Asn Leu 15 100 9 PRT Artificial CLP-2510 100 Phe Asn Leu Pro Pro His Phe Ser Ala1 5 101 9 PRT Artificial CLP-2511 101 Asn Leu Pro Pro His Phe Ser AlaVal 1 5 102 9 PRT Artificial CLP-2512 102 Ser Ala Val Gly Ser Asp AsnAsp Ile 1 5 103 9 PRT Artificial CLP-2513 103 Lys Asn Glu Gly Pro LeuAsn Val Val 1 5 104 9 PRT Artificial CLP-2514 104 Thr Lys Cys Val HisCys Gly Ile Val 1 5 105 9 PRT Artificial CLP-2515 105 Cys Val His CysGly Ile Val Phe Leu 1 5 106 9 PRT Artificial CLP-2516 106 Cys Gly IleVal Phe Leu Asp Glu Val 1 5 107 9 PRT Artificial CLP-2517 107 Phe LeuAsp Glu Val Met Tyr Ala Leu 1 5 108 9 PRT Artificial CLP-2518 108 ValMet Tyr Ala Leu His Met Ser Cys 1 5 109 9 PRT Artificial CLP-2519 109Phe Gln Cys Ser Ile Cys Gln His Leu 1 5 110 9 PRT Artificial CLP-2520110 Gly Leu His Arg Asn Asn Ala Gln Val 1 5 111 9 PRT ArtificialCLP-2477 111 Lys Met Gly Glu Pro Val Ser Glu Ser 1 5 112 9 PRTArtificial CLP-2478 112 Gly Leu Lys Glu Lys Val Trp Thr Glu 1 5 113 39DNA Artificial Primer, 9616SXC 113 cagtacggat ccaccatggc cgagctgcgcctgaagggc 39 114 38 DNA Artificial Primer, 9617SXC 114 ccacgaggatccttaggaga atattcggat ggcttgcg 38 115 20 DNA Artificial Primer, 9620MC115 taatacgact cactataggg 20 116 18 DNA Artificial Primer, 9621MC 116tagaaggcac agtcgagg 18 117 23 DNA Artificial Primer, 9618MC 117gaaaacgact tcctggcggg gag 23 118 22 DNA Artificial Primer, 9619MC 118gctcacccag gcgtggggcc tc 22 119 9 PRT Artificial CLP-2599 119 Val ProVal Pro Thr Ser Glu His Val 1 5 120 9 PRT Artificial CLP-2602 120 ProThr Ser Glu His Val Ala Glu Ile 1 5 121 9 PRT Artificial CLP-2609 121Glu Ile Val Gly Arg Gln Cys Lys Ile 1 5 122 9 PRT Artificial CLP-2616122 Lys Ile Lys Ala Leu Arg Ala Lys Thr 1 5 123 9 PRT ArtificialCLP-2618 123 Lys Ala Leu Arg Ala Lys Thr Asn Thr 1 5 124 9 PRTArtificial CLP-2619 124 Ala Leu Arg Ala Lys Thr Asn Thr Tyr 1 5 125 9PRT Artificial CLP-2620 125 Leu Arg Ala Lys Thr Asn Thr Tyr Ile 1 5 1269 PRT Artificial CLP-2624 126 Thr Asn Thr Tyr Ile Lys Thr Pro Val 1 5127 9 PRT Artificial CLP-2627 127 Tyr Ile Lys Thr Pro Val Arg Gly Glu 15 128 9 PRT Artificial CLP-2630 128 Thr Pro Val Arg Gly Glu Glu Pro Val1 5 129 9 PRT Artificial CLP-2633 129 Arg Gly Glu Glu Pro Val Phe MetVal 1 5 130 9 PRT Artificial CLP-2640 130 Met Val Thr Gly Arg Arg GluAsp Val 1 5 131 9 PRT Artificial CLP-2641 131 Val Thr Gly Arg Arg GluAsp Val Ala 1 5 132 9 PRT Artificial CLP-2643 132 Gly Arg Arg Glu AspVal Ala Thr Ala 1 5 133 9 PRT Artificial CLP-2647 133 Asp Val Ala ThrAla Arg Arg Glu Ile 1 5 134 9 PRT Artificial CLP-2648 134 Val Ala ThrAla Arg Arg Glu Ile Ile 1 5 135 9 PRT Artificial CLP-2650 135 Thr AlaArg Arg Glu Ile Ile Ser Ala 1 5 136 9 PRT Artificial CLP-2651 136 AlaArg Arg Glu Ile Ile Ser Ala Ala 1 5 137 9 PRT Artificial CLP-2655 137Ile Ile Ser Ala Ala Glu His Phe Ser 1 5 138 9 PRT Artificial CLP-2656138 Ile Ser Ala Ala Glu His Phe Ser Met 1 5 139 9 PRT ArtificialCLP-2657 139 Ser Ala Ala Glu His Phe Ser Met Ile 1 5 140 9 PRTArtificial CLP-2659 140 Ala Glu His Phe Ser Met Ile Arg Ala 1 5 141 9PRT Artificial CLP-2663 141 Ser Met Ile Arg Ala Ser Arg Asn Lys 1 5 1429 PRT Artificial CLP-2666 142 Arg Ala Ser Arg Asn Lys Ser Gly Ala 1 5143 9 PRT Artificial CLP-2670 143 Asn Lys Ser Gly Ala Ala Phe Gly Val 15 144 9 PRT Artificial CLP-2673 144 Gly Ala Ala Phe Gly Val Ala Pro Ala1 5 145 9 PRT Artificial CLP-2674 145 Ala Ala Phe Gly Val Ala Pro AlaLeu 1 5 146 9 PRT Artificial CLP-2677 146 Gly Val Ala Pro Ala Leu ProGly Gln 1 5 147 9 PRT Artificial CLP-2678 147 Val Ala Pro Ala Leu ProGly Gln Val 1 5 148 9 PRT Artificial CLP-2680 148 Pro Ala Leu Pro GlyGln Val Thr Ile 1 5 149 9 PRT Artificial CLP-2681 149 Ala Leu Pro GlyGln Val Thr Ile Arg 1 5 150 9 PRT Artificial CLP-2682 150 Leu Pro GlyGln Val Thr Ile Arg Val 1 5 151 9 PRT Artificial CLP-2684 151 Gly GlnVal Thr Ile Arg Val Arg Val 1 5 152 9 PRT Artificial CLP-2689 152 ArgVal Arg Val Pro Tyr Arg Val Val 1 5 153 9 PRT Artificial CLP-2691 153Arg Val Pro Tyr Arg Val Val Gly Leu 1 5 154 9 PRT Artificial CLP-2692154 Val Pro Tyr Arg Val Val Gly Leu Val 1 5 155 9 PRT ArtificialCLP-2695 155 Arg Val Val Gly Leu Val Val Gly Pro 1 5 156 9 PRTArtificial CLP-2698 156 Gly Leu Val Val Gly Pro Lys Gly Ala 1 5 157 9PRT Artificial CLP-2699 157 Leu Val Val Gly Pro Lys Gly Ala Thr 1 5 1589 PRT Artificial CLP-2700 158 Val Val Gly Pro Lys Gly Ala Thr Ile 1 5159 9 PRT Artificial CLP-2710 159 Arg Ile Gln Gln Gln Thr Asn Thr Tyr 15 160 9 PRT Artificial CLP-2711 160 Ile Gln Gln Gln Thr Asn Thr Tyr Ile1 5 161 9 PRT Artificial CLP-2712 161 Gln Gln Gln Thr Asn Thr Tyr IleIle 1 5 162 9 PRT Artificial CLP-2713 162 Gln Gln Thr Asn Thr Tyr IleIle Thr 1 5 163 9 PRT Artificial CLP-2718 163 Tyr Ile Ile Thr Pro SerArg Asp Arg 1 5 164 9 PRT Artificial CLP-2721 164 Thr Pro Ser Arg AspArg Asp Pro Val 1 5 165 9 PRT Artificial CLP-2724 165 Arg Asp Arg AspPro Val Phe Glu Ile 1 5 166 9 PRT Artificial CLP-2731 166 Glu Ile ThrGly Ala Pro Gly Asn Val 1 5 167 9 PRT Artificial CLP-2734 167 Gly AlaPro Gly Asn Val Glu Arg Ala 1 5 168 9 PRT Artificial CLP-2738 168 AsnVal Glu Arg Ala Arg Glu Glu Ile 1 5 169 9 PRT Artificial CLP-2744 169Glu Glu Ile Glu Thr His Ile Ala Val 1 5 170 9 PRT Artificial CLP-2746170 Ile Glu Thr His Ile Ala Val Arg Thr 1 5 171 9 PRT ArtificialCLP-2749 171 His Ile Ala Val Arg Thr Gly Lys Ile 1 5 172 9 PRTArtificial CLP-2750 172 Ile Ala Val Arg Thr Gly Lys Ile Leu 1 5 173 9PRT Artificial CLP-2756 173 Lys Ile Leu Glu Tyr Asn Asn Glu Asn 1 5 1749 PRT Artificial CLP-2760 174 Tyr Asn Asn Glu Asn Asp Phe Leu Ala 1 5175 9 PRT Artificial CLP-2762 175 Asn Glu Asn Asp Phe Leu Ala Gly Ser 15 176 9 PRT Artificial CLP-2766 176 Phe Leu Ala Gly Ser Pro Asp Ala Ala1 5 177 9 PRT Artificial CLP-2767 177 Leu Ala Gly Ser Pro Asp Ala AlaIle 1 5 178 9 PRT Artificial CLP-2774 178 Ala Ile Asp Ser Arg Tyr SerAsp Ala 1 5 179 9 PRT Artificial CLP-2777 179 Ser Arg Tyr Ser Asp AlaTrp Arg Val 1 5 180 9 PRT Artificial CLP-2785 180 Val His Gln Pro GlyCys Lys Pro Leu 1 5 181 9 PRT Artificial CLP-2793 181 Leu Ser Thr PheArg Gln Asn Ser Leu 1 5 182 9 PRT Artificial CLP-2801 182 Leu Gly CysIle Gly Glu Cys Gly Val 1 5 183 9 PRT Artificial CLP-2807 183 Cys GlyVal Asp Ser Gly Phe Glu Ala 1 5 184 9 PRT Artificial CLP-2812 184 GlyPhe Glu Ala Pro Arg Leu Asp Val 1 5 185 9 PRT Artificial CLP-2817 185Arg Leu Asp Val Tyr Tyr Gly Val Ala 1 5 186 9 PRT Artificial CLP-2819186 Asp Val Tyr Tyr Gly Val Ala Glu Thr 1 5 187 9 PRT ArtificialCLP-2823 187 Gly Val Ala Glu Thr Ser Pro Pro Leu 1 5 188 9 PRTArtificial CLP-2825 188 Ala Glu Thr Ser Pro Pro Leu Trp Ala 1 5 189 9PRT Artificial CLP-2830 189 Pro Leu Trp Ala Gly Gln Glu Asn Ala 1 5 1909 PRT Artificial CLP-2833 190 Ala Gly Gln Glu Asn Ala Thr Pro Thr 1 5191 9 PRT Artificial CLP-2835 191 Gln Glu Asn Ala Thr Pro Thr Ser Val 15 192 9 PRT Artificial CLP-2843 192 Val Leu Phe Ser Ser Ala Ser Ser Ser1 5 193 9 PRT Artificial CLP-2857 193 Lys Ala Arg Ala Gly Pro Pro GlyAla 1 5 194 9 PRT Artificial CLP-2869 194 Pro Ala Thr Ser Ala Gly ProGlu Leu 1 5 195 9 PRT Artificial CLP-2870 195 Ala Thr Ser Ala Gly ProGlu Leu Ala 1 5 196 9 PRT Artificial CLP-2872 196 Ser Ala Gly Pro GluLeu Ala Gly Leu 1 5 197 9 PRT Artificial CLP-2879 197 Gly Leu Pro ArgArg Pro Pro Gly Glu 1 5 198 9 PRT Artificial CLP-2887 198 Glu Pro LeuGln Gly Phe Ser Lys Leu 1 5 199 9 PRT Artificial CLP-2892 199 Phe SerLys Leu Gly Gly Gly Gly Leu 1 5 200 9 PRT Artificial CLP-2894 200 LysLeu Gly Gly Gly Gly Leu Arg Ser 1 5 201 9 PRT Artificial CLP-2899 201Gly Leu Arg Ser Pro Gly Gly Gly Arg 1 5 202 9 PRT Artificial CLP-2909202 Cys Met Val Cys Phe Glu Ser Glu Val 1 5 203 9 PRT ArtificialCLP-2910 203 Met Val Cys Phe Glu Ser Glu Val Thr 1 5 204 9 PRTArtificial CLP-2911 204 Val Cys Phe Glu Ser Glu Val Thr Ala 1 5 205 9PRT Artificial CLP-2913 205 Phe Glu Ser Glu Val Thr Ala Ala Leu 1 5 2069 PRT Artificial CLP-2916 206 Glu Val Thr Ala Ala Leu Val Pro Cys 1 5207 9 PRT Artificial CLP-2917 207 Val Thr Ala Ala Leu Val Pro Cys Gly 15 208 9 PRT Artificial CLP-2920 208 Ala Leu Val Pro Cys Gly His Asn Leu1 5 209 9 PRT Artificial CLP-2921 209 Leu Val Pro Cys Gly His Asn LeuPhe 1 5 210 9 PRT Artificial CLP-2922 210 Val Pro Cys Gly His Asn LeuPhe Cys 1 5 211 9 PRT Artificial CLP-2927 211 Asn Leu Phe Cys Met GluCys Ala Val 1 5 212 9 PRT Artificial CLP-2929 212 Phe Cys Met Glu CysAla Val Arg Ile 1 5 213 9 PRT Artificial CLP-2933 213 Cys Ala Val ArgIle Cys Glu Arg Thr 1 5 214 9 PRT Artificial CLP-2936 214 Arg Ile CysGlu Arg Thr Asp Pro Glu 1 5 215 9 PRT Artificial CLP-2940 215 Arg ThrAsp Pro Glu Cys Pro Val Cys 1 5 216 9 PRT Artificial CLP-2945 216 CysPro Val Cys His Ile Thr Ala Thr 1 5 217 9 PRT Artificial CLP-2947 217Val Cys His Ile Thr Ala Thr Gln Ala 1 5 218 9 PRT Artificial CLP-2950218 Ile Thr Ala Thr Gln Ala Ile Arg Ile 1 5

What is claimed is:
 1. An expression vector comprising the nucleic acidsequence as illustrated in SEQ ID NO.: 1 or 3 or a fragment thereof. 2.The expression vector of claim 1 wherein the vector is a plasmid or aviral vector.
 3. The expression vector of claim 2 wherein the viralvector is selected from the group consisting of poxvirus, alphavirus,adenovirus, retrovirus, herpesvirus, and adeno-associated virus.
 4. Theexpression vector of claim 3 wherein the viral vector is a poxvirusselected from the group consisting of vaccinia, NYVAC, avipox,canarypox, ALVAC, ALVAC(2), fowlpox, and TROVAC.
 5. The expressionvector of claim 4 wherein the viral vector is a poxvirus selected fromthe group consisting of NYVAC, ALVAC, and ALVAC(2).
 6. The expressionvector of claim 1 further comprising at least one additionaltumor-associated antigen.
 7. The expression vector of claim 6 whereinthe vector is a plasmid or a viral vector.
 8. The expression vector ofclaim 7 wherein the viral vector is selected from the group consistingof poxvirus, alphavirus, adenovirus, retrovirus, herpesvirus, andadeno-associated virus.
 9. The expression vector of claim 8 wherein theviral vector is a poxvirus selected from the group consisting ofvaccinia, NYVAC, avipox, canarypox, ALVAC, ALVAC(2), fowlpox, andTROVAC.
 10. The expression vector of claim 9 wherein the viral vector isa poxvirus selected from the group consisting of NYVAC, ALVAC, andALVAC(2).
 11. The expression vector of claim 1 further comprising atleast one nucleic sequence encoding an angiogenesis-associated antigen.12. The expression vector of claim 11 wherein the vector is a plasmid ora viral vector.
 13. The expression vector of claim 12 wherein the viralvector is selected from the group consisting of poxvirus, alphavirus,adenovirus, retrovirus, herpesvirus, and adeno-associated virus.
 14. Theexpression vector of claim 13 wherein the viral vector is a poxvirusselected from the group consisting of vaccinia, NYVAC, avipox,canarypox, ALVAC, ALVAC(2), fowlpox, and TROVAC.
 15. The expressionvector of claim 14 wherein the viral vector is a poxvirus selected fromthe group consisting of NYVAC, ALVAC, and ALVAC(2).
 16. The expressionvector of claim 6 further comprising at least one nucleic sequenceencoding an angiogenesis-associated antigen.
 17. The expression vectorof claim 16 wherein the vector is a plasmid or a viral vector.
 18. Theexpression vector of claim 17 wherein the viral vector is selected fromthe group consisting of poxvirus, alphavirus, adenovirus, retrovirus,herpesvirus, and adeno-associated virus.
 19. The expression vector ofclaim 17 wherein the viral vector is a poxvirus selected from the groupconsisting of vaccinia, NYVAC, avipox, canarypox, ALVAC, ALVAC(2),fowlpox, and TROVAC.
 20. The poxvirus of claim 18 wherein the viralvector is a poxvirus selected from the group consisting of NYVAC, ALVAC,and ALVAC(2).
 21. The expression vector of claim 1, 6, 11 or 16 furthercomprising at least one nucleic acid sequence encoding a co-stimulatorycomponent.
 22. The expression vector of claim 22 wherein the vector is aplasmid or a viral vector.
 23. The expression vector of claim 23 whereinthe viral vector is selected from the group consisting of poxvirus,alphavirus, adenovirus, retrovirus, herpesvirus, and adeno-associatedvirus.
 24. The expression vector of claim 24 wherein the viral vector isa poxvirus selected from the group consisting of vaccinia, NYVAC,avipox, canarypox, ALVAC, ALVAC(2), fowlpox, and TROVAC.
 25. Thepoxvirus of claim 18 wherein the viral vector is a poxvirus selectedfrom the group consisting of NYVAC, ALVAC, and ALVAC(2).
 26. Acomposition comprising an expression vector in a pharmaceuticallyacceptable carrier, said vector comprising the nucleic acid sequenceshown in SEQ ID NO.: 1 or 3 or a fragment thereof.
 27. The expressionvector of claim 26 wherein the vector is a plasmid or a viral vector.28. The expression vector of claim 27 wherein the viral vector isselected from the group consisting of poxvirus, alphavirus, adenovirus,retrovirus, herpesvirus, and adeno-associated virus.
 29. The expressionvector of claim 28 wherein the viral vector is a poxvirus selected fromthe group consisting of vaccinia, NYVAC, avipox, canarypox, ALVAC,ALVAC(2), fowlpox, and TROVAC.
 30. The poxvirus of claim 29 wherein theviral vector is a poxvirus selected from the group consisting of NYVAC,ALVAC, and ALVAC(2).
 31. A method for preventing or treating cancercomprising administering to a host an expression vector comprising thenucleic acid sequence illustrated in SEQ ID NO.: 1 or 3 or a fragmentthereof.
 32. The expression vector of claim 31 wherein the vector is aplasmid or a viral vector.
 33. The expression vector of claim 32 whereinthe viral vector is selected from the group consisting of poxvirus,alphavirus, adenovirus, retrovirus, herpesvirus, and adeno-associatedvirus.
 34. The expression vector of claim 33 wherein the viral vector isa poxvirus selected from the group consisting of vaccinia, NYVAC,avipox, canarypox, ALVAC, ALVAC(2), fowlpox, and TROVAC.
 35. Thepoxvirus of claim 34 wherein the viral vector is a poxvirus selectedfrom the group consisting of NYVAC, ALVAC, and ALVAC(2).
 36. A peptidederived from BFA4 as shown in Table V, VI or VII.
 37. A method forimmunizing a host against the tumor antigen BFA4 comprisingadministering to the patient a peptide shown in Table V, VI or VII,either alone or in combination with another agent, where the individualcomponents of the combination are administered simultaneously orseparately from one another.
 38. A peptide derived from BCY1 as shown inTable VIII or IX.
 39. A method for immunizing a host against the tumorantigen BCY1 comprising administering to the patient a peptide shown inTable VIII or IX, either alone or in combination with at least one otheragent, where the individual components of the combination areadministered simultaneously or separately from one another.